Combination cancer therapy using chimeric antigen receptor engineered natural killer cells as chemotherapeutic drug carriers

ABSTRACT

Compositions are provided including NK cells that express chimeric antigen receptors (CARs) specific to CD19 and Her2 and a plurality of cell surface-bound multilamellar liposomal vesicles loaded with one or more anti-cancer therapeutics at an effective amount for inhibiting or killing tumor cells without causing toxicity to the NK cells. Methods of using these compositions to treat a subject with tumor are also provided, including administering an effective amount of the CAR-engineered NK cells, where an effective amount of anti-tumor therapeutics are delivered in particles (e.g., crosslinked multilamellar liposomal vesicles) that are bound to the surface of these CAR-engineered NK cells, without causing toxicity to the carrier NK cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalApplication No. 62/523,401, filed on Jun. 22, 2017, the disclosure ofwhich is hereby incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. AI068978awarded by National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

Composition and methods for treating cancer are described herein.

BACKGROUND OF THE INVENTION

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

The therapeutic limitations of conventional chemotherapeutic drugsinclude chemo-resistance, tumor recurrence, and metastasis. Numerousnanoparticle-based active targeting approaches have emerged to enhancethe intracellular concentration of drugs in tumor cells. However,efficient delivery of these systems to the tumor site while sparinghealthy tissue remains elusive.

Recently, much attention has been given to human immune cell-directednanoparticle drug delivery, as immune cells can traffic to the tumor andinflammatory sites. Natural killer cells are a subset of cytotoxiclymphocytes that play an important role in cancer immunosurveillance.Engineering of the human natural killer cell line, NK92, to expresschimeric antigen receptors to redirect their antitumor specificity hasshown significant promise.

Therefore it is an objective of the present invention to provide acomposition and/or a delivery system that combines cell-basedimmunotherapy and chemotherapeutics for enhanced delivery, efficacy andspecificity of anti-cancer therapies.

It is another objective of the present invention to provide a method oftreating a subject with tumor by utilizing both immunotherapy andchemotherapeutics

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, compositions and methods whichare meant to be exemplary and illustrative, not limiting in scope.

A chimeric antigen receptor (CAR)-engineered immune effector cell isprovided to improve tumor-targeted delivery and efficacy, where the CARcontains an extracellular antigen specific domain and the cell surfaceis bound with a plurality of nano- or microparticles that contain aneffective amount of active agents (e.g., chemotherapeutics) for efficacyagainst target cells without cytotoxicity to the carrier, CAR-engineeredimmune effector cell. Various embodiments provide that the immuneeffector cell is a natural killer (NK) cell. A CAR-engineered NK cellshave polynucleotides encoding chimeric antigen receptors (CARs) orhaving expressed on the surface CARs. Generally, the bound particles arenot endocytosed or internalized by the CAR-engineered NK cell, eventhough NK cells have phagocytotic capabilities. In some aspects, theCAR-engineered NK cells carry an effective amount of active agents(e.g., chemotherapeutics) to kill target cells without succumbing tochemotherapeutics-induced toxicity themselves. For example, an averageeffective amount of anti-tumor therapeutics that are delivered perCAR-engineered NK cell results in inhibition or killing of at least 10%,20%, 30%, 40%, 50%, or 60% of targeted antigen-expressing tumor cells ata cell number ratio between 1:1 and 10:1 (e.g., 1:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1 or 10:1) of engineered immune effectorcell:antigen-expressing target tumor cell, but does not cause more than1%, 3%, 5%, 7%, 10% or 15% cell death or cytotoxicity to theCAR-engineered NK cells. Other embodiments provide that the cell numberratio of engineered immune effector cell:antigen-expressing target tumorcell is greater than 10:1, e.g., 11:1, 12:1, 13:1, 14:1, 15:1, 20:125:1; which results in inhibition or killing of at least 10%, 20%, 30%,40%, 50%, or 60% of targeted antigen-expressing tumor cells. Generally,in in vivo models, the dosage required of chemotherapeutic agentsdelivered via nanoparticles conjugated to the surface of NK cells toachieve inhibition of tumor growth and reduction of tumor size may be atleast 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold,15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold or 200-fold lessthan the dosage required of free chemotherapeutic agents (without NKcells) for similar inhibition or reduction efficacy.

In various embodiments, the CAR-engineered NK cells having bound on thesurface a plurality of active agent-loaded particles accumulate and havea higher concentration in the tumor environment followingadministration, and enhance antitumor efficacy, compared to that ofCAR-engineered NK cells lacking surface-bound, active agent-loadedparticles, whether these cells are administered alone or in a mixturewith free, unbound active agent-loaded particles.

The conjugation of particles to the surface of CAR-engineered NK cellsincreases the release of type II interferon (IFN-γ) when the cells arecultured with target cells that have the cognate antigen (which the CARrecognizes), compared with the CAR-engineered NK cells that are culturedwith target cells without the antigen. The released IFN-γ sensitizestumor cells to NK cytotoxicity and initiates broad adaptive and innateimmune responses.

In some embodiments, CAR-engineered NK92 cells are preferred toCAR-engineered T cells. NK92 cells proliferate in shorter time span thanT cells, and are identical to parental cell line, thereby minimizingproblems with donor variability. NK92 cells after irradiation aregenerally safe to use clinically, decreasing the risk of off-targeteffects compared to CAR-engineered T cells. Allogenic NK92 cell-basedtherapy including CAR engineering and particle conjugation, as disclosedherein, is generally less expensive than autologous CAR-engineering Tcell-based similar therapy.

In one embodiment, the CAR binds to CD19. In another embodiment, the CARbinds Her2. In a further embodiment, the cell comprises bispecific CARsthat bind CD19 and Her2. In some embodiments, the cells comprise thenucleic acids wherein the nucleic acids encode CARs that bind CD19 andHer2.

In some embodiments, the particles bound to the surface ofCAR-engineered NK cells are nanoparticles, having an averaged diameterbetween 1 nm and 1,000 nm. In other embodiments, the particles bound tothe surface of CAR-engineered NK cells are microparticles. Exemplaryparticles include liposomes or alternative liposomal formulations, suchas cross-linked multilamellar liposomes (cMLV), and controlled releasepolymeric nanoparticles. In some aspects, cMLVs, as the active agentcarrier, are bound to the surface of CAR-expressing NK cells, whereinterlipid bilayers are crosslinked in a liposome, resulting in a robustmultilamellar structure. In other aspects, polymeric nanoparticles arethe active agent carrier and bound to the surface of CAR-expressing NKcells. Depending on the solubility of the incorporated active agent,hydrophobic polymers or block copolymers may be selected, e.g.,poly(lactic acid), poly(glycolic acid) or copolymer thereof, to formnanoparticles for controlled released of active agent therefrom. In oneembodiment, cMLV are incubated with CAR-engineered NK cells at a numberratio greater than 500:1, e.g., about 1,000:1 or 2,000:1, to result in aconjugation ratio of about 100-150 cMLVs per CAR-engineered NK cell. Inother embodiments depending on the size, chemical composition andlinkage functional groups, the number of conjugated nanoparticles perCAR-engineered NK cell is between 400 and 350, between 350 and 300,between 300 and 250, between 250 and 200, between 200 and 150, orbetween 150 and 100. An exemplary conjugation chemistry is betweenmaleimide group functionalized on the particles and free thiols on theimmune effector cells. Optionally, a linker between the particles andthe cell surface is present, e.g., via a polyethylene glycol.

Exemplary active agents include tumor therapeutics, such as paclitaxeland SN-38, pro-inflammatory cytokines, such as interleukin (IL)-15 andIL-21, check point inhibitors (e.g., PD-1 inhibitor including antibodiesto PD-1), and immune-modulating agents. In one embodiment, thechemotherapeutic agent is paclitaxel. In another embodiment, two or morechemotherapeutic agents, such as paclitaxel and doxorubicin, aredelivered in the same or individual nanoparticles that are bound to thesurface of one CAR-expressing NK cell.

Also provided are pharmaceutical compositions comprising a NK cellcontaining nucleic acids encoding a chimeric antigen receptor (CAR),wherein the cell further contains on the surface bound crosslinkedmultilamellar liposomal vesicles (cMLVs) that encapsulate achemotherapeutic agent; and a pharmaceutically acceptable carrier.

Further provided herein are methods for treating cancer in a subject inneed thereof comprising administering to the subject an effective amountof the cells and/or pharmaceutically composition, as described herein.

A method of treating a subject with tumor(s) is also provided, wherein acomposition including CAR-engineered NK cells (e.g., NK92 cells) withsurface-bound nanoparticles is administered to the subject, thenanoparticles containing anti-cancer therapeutics, so as to enhance thedelivery and efficacy of therapeutics and reduce off-target toxicity tonormal tissue. Generally, the CAR is designed to bind an antigen of thecancer cells of the subject to which the composition is administered.For example, anti-CD19 CAR, anti-Her2 CAR, or both are expressed in theNK cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1A-FIG. 1D depict NK92 cell conjugation to maleimide-functionalizedcMLVs. FIG. 1A shows a schematic of CAR.NK cells conjugated toPTX-loaded cMLVs. CARs are derived from the single chain variablefragment (scFv) of an antibody and the T cell receptor signalingcomplex. CARs can be transduced into NK92 cells and cMLVs can conjugateto the cell surface by interacting with free thiols. FIG. 1B shows cMLVsconjugated to the NK cell surface at various cMLV: cell ratios. cMLVscontaining the fluorescent dye DiD were co-incubated with NK cells overa range of number ratios. The number of cMLVs on the surface of eachcell was calculated by analyzing the DiD fluorescence. The ratio of1000:1 provided the maximum amount of cMLVs per cell and was used infuture experiments. FIG. 1C shows confocal microscopic images of CAR.NKcells conjugated to DiD-loaded cMLVs (cMLV(DiD)). CAR.NK cells werelabeled with 1 μM CFSE and washed with PBS prior to conjugation tocMLV(DiD). Confocal microscopy was used to visualize the cMLVs on theCAR.NK cell surface. FIG. 1D shows an internalization assay ofconjugated cMLVs. CAR.NK cells were conjugated withcarboxyfluorescein-tagged maleimide-labeled cMLVs. The extracellularconjugation was quenched by trypan blue to differentiate surface-boundand internalized cMLVs 2 hours after conjugation. Attachment of cMLVs toCAR.NK-cells did not trigger the internalization of particles by thecells. Summarized statistics are displayed in the graphs (n=3, mean±SEM;NS, not significant; *p<0.05; **p<0.01; ***p<0.001).

FIG. 1E depicts CAR expression in transduced NK cells. Non-transduced NKcells were used as a negative control (gray shaded peaks). Anti-CD19CARs were detected using flow cytometry after being labeled withbiotinylated Protein L and streptavidin conjugated to FITC. Anti-Her2CAR.NK cells were detected with flow cytometry after being labeled withrhHer2-Fc chimera and PE-labeled goat anti-human Fc.

FIG. 1F depicts confocal microscopy of CAR.NK cells conjugated toDiD-loaded cMLVs (cMLV(DiD))-3D and Z-stacked images. CAR.NK cells werelabeled with 1 μM CFSE and washed with PBS prior to conjugation tocMLV(DiD).

FIG. 2A-FIG. 2B depict cytotoxicity of CAR.NK cells against CD19⁺ orHer2⁺ target cells. FIG. 2A shows cytotoxicity of anti-CD19 CAR.NKcells. Anti-CD19 CAR.NK cells were co-cultured with CD19⁻ SKOV3 cells orCD19⁺ SKOV.CD19 cells for 24 hours at 1:1, 5:1, or 10:1effector-to-target ratios and cytotoxicity was measured. FIG. 2B showscytotoxicity of anti-Her2 CAR.NK cells. Anti-Her2 CAR.NK cells wereco-cultured with Her2⁻ MDA.MB.468 cells or Her2⁺ SKOV3 cells for 24hours at 1:1, 5:1, or 10:1 effector-to-target ratios and cytotoxicitywas measured. Summarized statistics are displayed in the graphs (n=3,mean±SEM; NS, not significant; *p<0.05; **p<0.01; ***p<0.001).

FIG. 2C depicts cytotoxicity comparison between irradiated (5 Gy) andnonirradiated CAR.NK cells. NK or CAR.NK cells were cocultured withSKOV.CD19 cells for 24 hours at 1:1, 5:1, or 10:1 effector-to-targetratios and cytotoxicity was measured.

FIG. 2D depicts cell viability assay with NK and SKOV3 cells exposed toPTX. Cells were incubated with various concentrations of cMLV(PTX). Cellviability percentage was determined by subtracting absorbance valuesobtained from media-only wells from the treated wells and thennormalized by the control wells containing cells without drugs.

FIG. 2E depicts PTX release kinetics from free cMLVs and CAR.NK.cMLVs.To obtain the release kinetics of PTX from cMLVs before and after cellconjugation, cMLV(PTX) and CAR.NK.cMLV(PTX) were incubated in 10%FBS-containing media at 37° C. and were spun down and resuspended withfresh media daily. The PTX was quantified from the removed media by HPLCevery day.

FIG. 3A-FIG. 3E depict CAR.NK cytokine release and migration whenconjugated to cMLVs. FIG. 3A and FIG. 3B show IFNγ staining assays.Anti-CD19 (FIG. 3A) or anti-Her2 (FIG. 3B) CAR.NK cells were coculturedwith various target cells with Brefeldin A protein transport inhibitorfor 6 hours to detect IFNγ release. Unstimulated CAR.NK cells served asa negative control. CAR.NK cells were either unconjugated or conjugatedwith empty cMLVs (CAR.NK.cMLV(EMPTY)) or PTX-loaded cMLVs(CAR.NK.cMLV(PTX)). IFNγ was measured with intracellular staining. FIG.3C and FIG. 3D show cytotoxicity assays. Anti-CD19 (FIG. 3C) oranti-Her2 (FIG. 3D) CAR.NK cells were cocultured with various targetcells at a 1:1 ratio for 24 hours and cytotoxicity was measured. CAR.NKcells were either unconjugated or conjugated with empty cMLVs(CAR.NK.cMLV(EMPTY)) or PTX-loaded cMLVs (CAR.NK.cMLV(PTX)). FIG. 3Eshows the migration assay. Unconjugated NK or NK conjugated tocMLV(EMPTY) were plated in the upper chambers of a Transwell plate.Negative controls had plain media in the lower wells, and CXCL9 was usedas a chemoattractant in the lower wells of non-control groups. After 6hours of incubation, media from the lower chambers was collected and NKcells were counted. Summarized statistics are displayed in the graphs(n=3, mean±SEM; NS, not significant; *p<0.05; **p<0.01; ***p<0.001).

FIG. 4A-FIG. 4F depict biodistribution of free cMLV(DiD) and conjugatedCAR.NK.cMLV(DiD). Biodistribution data 24 hours (FIGS. 4A and 4B), 48hours (FIGS. 4C and 4D), or 72 hours (FIGS. 4E and 4F) after intravenousinjections. NOD/scid/IL2rγ−/− (NSG) mice bearing subcutaneous SKOV3.CD19tumors were intravenously injected with 2×10⁷ CAR.NK cells conjugatedwith DiD-labeled cMLVs or an equivalent number of DiD-labeled cMLVsalone (n=3 per group per time point). After 24 hours, 48 hours, and 72hours, indicated tissues were removed, weighed, and macerated withscissors. Specific DiD tissue fluorescence for each organ was quantifiedusing the IVIS Spectrum imaging system and the mean percentage ofinjected dose per gram of tissue (% ID/g) was calculated as the finalreadout. Summarized statistics are displayed in the graphs (n=3,mean±SEM; NS, not significant; *p<0.05; **p<0.01; ***p<0.001).

FIG. 5 depicts antitumor efficacy of CAR.NK.cMLV(PTX) in solid tumorxenograft model. Tumor growth curve. SKOV.CD19 cells were injectedsubcutaneously into the right flank of NSG mice on Day 0. Mice wererandomized into six groups (n=5 per group) and treated according totheir group description four times total, 3-4 days apart via tail veininjection. Tumor size was measured with a fine caliper (n=5, mean±SEM;NS, not significant; *p<0.05; **p<0.01; ***p<0.001).

FIG. 6A-FIG. 6C depict ex vivo analysis of CAR.NK.cMLV(PTX) treatment.FIG. 6A shows the intratumoral PTX concentration. Thawed tumor sampleswere homogenized and PTX concentrations analyzed using HPLC (n=3,mean±SEM; NS, not significant; *p<0.05; **p<0.01; ***p<0.001). FIG. 6Bshows TUNEL assay of fixed frozen tumor sections. Tumor sections werestained with a TUNEL kit according to the manufacturer's instructionsand imaged with confocal microscopy. Representative images are shownherein. FIG. 6C shows the histology analysis for cardiac toxicity.Cardiac tissue was fixed and frozen, and sections were mounted on glassslides. The frozen sections were stained with hematoxylin and eosin.Histopathologic specimens were examined by light microscopy.Representative images are shown herein.

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Allen et al., Remington: The Science and Practice of Pharmacy22^(nd) ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al.,Introduction to Nanoscience and Nanotechnology, CRC Press (2008);Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology 3^(rd) ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006);Smith, March's Advanced Organic Chemistry Reactions, Mechanisms andStructure 7^(th) ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton,Dictionary of DNA and Genome Technology 3^(rd) ed., Wiley-Blackwell(Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A LaboratoryManual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor,N.Y. 2012), provide one skilled in the art with a general guide to manyof the terms used in the present application. For references on how toprepare antibodies, see Greenfield, Antibodies A Laboratory Manual2^(nd) ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013);Köhler and Milstein, Derivation of specific antibody-producing tissueculture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July,6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No.5,585,089 (1996 December); and Riechmann et al., Reshaping humanantibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Other features and advantages of theinvention will become apparent from the following detailed description,taken in conjunction with the accompanying drawings, which illustrate,by way of example, various features of embodiments of the invention.Indeed, the present invention is in no way limited to the methods andmaterials described. For convenience, certain terms employed herein, inthe specification, examples and appended claims are collected here.

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments, and are not intended to limit theclaimed invention, because the scope of the invention is limited only bythe claims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areuseful to an embodiment, yet open to the inclusion of unspecifiedelements, whether useful or not. It will be understood by those withinthe art that, in general, terms used herein are generally intended as“open” terms (e.g., the term “including” should be interpreted as“including but not limited to,” the term “having” should be interpretedas “having at least,” the term “includes” should be interpreted as“includes but is not limited to,” etc.).

Unless stated otherwise, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe application (especially in the context of claims) can be construedto cover both the singular and the plural. The recitation of ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate value falling within the range.Unless otherwise indicated herein, each individual value is incorporatedinto the specification as if it were individually recited herein. Allmethods described herein can be performed in any suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context.The use of any and all examples, or exemplary language (for example,“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the application and does not pose alimitation on the scope of the application otherwise claimed. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.” No language in thespecification should be construed as indicating any non-claimed elementessential to the practice of the application.

As used herein, the term “about” refers to a measurable value such as anamount, a time duration, and the like, and encompasses variations of±20%, ±10%, ±5%, ±1%, ±0.5% or ±0.1% from the specified value.

“Chimeric antigen receptor” or “CAR” or “CARs” as used herein refers toengineered receptors, which graft an antigen specificity onto cells (forexample NK cells). CARs are also known as artificial T-cell receptors,chimeric T-cell receptors or chimeric immunoreceptors. In variousembodiments, CARs are recombinant polypeptides comprising anantigen-specific domain (ASD), a hinge region (HR), a transmembranedomain (TMD), co-stimulatory domain (CSD) and an intracellular signalingdomain (ISD).

“Effector function” refers to the specialized function of adifferentiated cell. Effector function of a T-cell, for example, may becytolytic/cytotoxicity activity or helper activity including thesecretion of cytokines.

“Disease targeted by genetically modified cells” as used hereinencompasses the targeting of any cell involved in any manner in anydisease by the genetically modified cells of the invention, irrespectiveof whether the genetically modified cells target diseased cells orhealthy cells to effectuate a therapeutically beneficial result. Thegenetically modified cells include but are not limited to geneticallymodified T-cells, NK cells, hematopoietic stem cells, pluripotentembryonic stem cells or embryonic stem cells. The genetically modifiedcells described herein express CARs that target specific antigens and incombination, function as chemotherapeutic drug delivery carriers.Examples of antigens which may be targeted include but are not limitedto antigens expressed on B-cells; antigens expressed on carcinomas,sarcomas, lymphomas, leukemia, germ cell tumors, and blastomas; antigensexpressed on various immune cells; and antigens expressed on cellsassociated with various hematologic diseases, autoimmune diseases,and/or inflammatory diseases. Other antigens that may be targeted willbe apparent to those of skill in the art and may be targeted by the CARsof the invention in connection with alternate embodiments thereof.

“Autologous” cells as used herein refers to cells derived from the sameindividual as to whom the cells are later to be re-administered into.

“Genetically modified cells”, “redirected cells”, “geneticallyengineered cells”, “engineered cells” or “modified cells” as used hereinrefer to cells that express antigen-specific CARs and further haveparticles, preferably nanoparticles such as nano-sized liposomes (suchas multilamellar liposomal vesicles) that carry a therapeutic agent suchas a chemotherapeutic agent, where the particles are bound to thesurface of the cells. In some embodiments, the genetically modifiedcells express CARs that target specific antigens and in combination,function as chemotherapeutic drug delivery carriers.

“Immune cell” as used herein refers to the cells of the mammalian immunesystem including but not limited to antigen presenting cells, B-cells,basophils, cytotoxic T-cells, dendritic cells, eosinophils,granulocytes, helper T-cells, leukocytes, lymphocytes, macrophages, mastcells, memory cells, monocytes, natural killer cells, neutrophils,phagocytes, plasma cells and T-cells.

The term “immune effector function” of the CAR-containing cell refers toany of the activities shown by the CAR-expressing cell upon stimulationby a stimulatory molecule. Examples of immune effector function, e.g.,in a CAR-T cell, include cytolytic activity and helper activity,including the secretion of cytokines.

“Immune effector cell” as used herein includes the T cells and naturalkiller (NK) cells.

“Immune response” as used herein refers to immunities including but notlimited to innate immunity, humoral immunity, cellular immunity,immunity, inflammatory response, acquired (adaptive) immunity,autoimmunity and/or overactive immunity.

As used herein, “CD4 lymphocytes” refer to lymphocytes that express CD4,i.e., lymphocytes that are CD4+. CD4 lymphocytes may be T cells thatexpress CD4.

The terms “T-cell” and “T-lymphocyte” are interchangeable and usedsynonymously herein. Examples include but are not limited to naïve Tcells, central memory T cells, effector memory T cells or combinationsthereof.

As used herein, the term “antibody” refers to an intact immunoglobulinor to a monoclonal or polyclonal antigen-binding fragment with the Fc(crystallizable fragment) region or FcRn binding fragment of the Fcregion, referred to herein as the “Fc fragment” or “Fc domain”.Antigen-binding fragments may be produced by recombinant DNA techniquesor by enzymatic or chemical cleavage of intact antibodies.Antigen-binding fragments include, inter alia, Fab, Fab′, F(ab′)2, Fv,dAb, and complementarity determining region (CDR) fragments,single-chain antibodies (scFv), single domain antibodies, chimericantibodies, diabodies and polypeptides that contain at least a portionof an immunoglobulin that is sufficient to confer specific antigenbinding to the polypeptide. The Fc domain includes portions of two heavychains contributing to two or three classes of the antibody. The Fcdomain may be produced by recombinant DNA techniques or by enzymatic(e.g. papain cleavage) or via chemical cleavage of intact antibodies.

The term “antibody fragment,” as used herein, refer to a proteinfragment that comprises only a portion of an intact antibody, generallyincluding an antigen binding site of the intact antibody and thusretaining the ability to bind antigen. Examples of antibody fragmentsencompassed by the present definition include: (i) the Fab fragment,having VL, CL, VH and CH1 domains; (ii) the Fab′ fragment, which is aFab fragment having one or more cysteine residues at the C-terminus ofthe CH1 domain; (iii) the Fd fragment having VH and CH1 domains; (iv)the Fd′ fragment having VH and CH1 domains and one or more cysteineresidues at the C-terminus of the CH1 domain; (v) the Fv fragment havingthe VL and VH domains of a single arm of an antibody; (vi) the dAbfragment (Ward et al., Nature 341, 544-546 (1989)) which consists of aVH domain; (vii) isolated CDR regions; (viii) F(ab′)2 fragments, abivalent fragment including two Fab′ fragments linked by a disulphidebridge at the hinge region; (ix) single chain antibody molecules (e.g.,single chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); andHuston et al., PNAS (USA) 85:5879-5883 (1988)); (x) “diabodies” with twoantigen binding sites, comprising a heavy chain variable domain (VH)connected to a light chain variable domain (VL) in the same polypeptidechain (see, e.g., EP 404,097; WO 93/11161; and Hollinger et al., Proc.Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi) “linear antibodies”comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, togetherwith complementary light chain polypeptides, form a pair of antigenbinding regions (Zapata et al. Protein Eng. 8(10):1057-1062 (1995); andU.S. Pat. No. 5,641,870).

“Single chain variable fragment”, “single-chain antibody variablefragments” or “scFv” antibodies as used herein refer to forms ofantibodies comprising the variable regions of only the heavy (V_(H)) andlight (V_(L)) chains, connected by a linker peptide. The scFvs arecapable of being expressed as a single chain polypeptide. The scFvsretain the specificity of the intact antibody from which it is derived.The light and heavy chains may be in any order, for example,V_(H)-linker-V_(L) or V_(L)-linker-V_(H), so long as the specificity ofthe scFv to the target antigen is retained.

“Complementarity determining region” (CDR) as used herein refers to theamino acid sequences within the variable regions of antibodies whichregions confer specificity and binding affinity. In general, there arethree CDRs in each of the light chain variable regions (LCDR1, LCDR2 andLCDR3) and three CDRs in each of the heavy chain variable regions (HCD1,HCDr2 and HCDR3). The boundaries of the CDRs may be determined usingmethods well known in the art including the “Kabat” numbering schemeand/or “Chothia” number scheme (Kabat et al. Sequences of Proteins ofImmunological Interest, 5^(th) Ed. Public Health Services, NationalInstitutes of Health, Bethesda, Md.; Al-Lazikani et al., (1997) JMB 273,927-948).

As used herein, the term “specific binding” means the contact between anantibody and an antigen with a binding affinity of at least 10⁻⁶ M. Incertain aspects, antibodies bind with affinities of at least about10⁻⁷M, and preferably 10⁻⁸M, 10⁻⁹ M, 10¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M.

“Therapeutic agents” as used herein refers to agents that are used to,for example, treat, inhibit, prevent, mitigate the effects of, reducethe severity of, reduce the likelihood of developing, slow theprogression of and/or cure, a disease. Diseases targeted by thetherapeutic agents include but are not limited to infectious diseases,cancers including but not limited to carcinomas, sarcomas, lymphomas,leukemia, germ cell tumors, and blastomas, antigens expressed on variousimmune cells, and antigens expressed on cells associated with varioushematologic diseases, and/or inflammatory diseases.

“Cancer” and “cancerous” refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. The term “cancer” is meant to include all types of cancerousgrowths or oncogenic processes, metastatic tissues or malignantlytransformed cells, tissues, or organs, irrespective of histopathologictype or stage of invasiveness. Examples of solid tumors includemalignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of thevarious organ systems, such as those affecting liver, lung, breast,lymphoid, gastrointestinal (e.g., colon), genitourinary tract (e.g.,renal, urothelial cells), prostate and pharynx. Adenocarcinomas includemalignancies such as most colon cancers, rectal cancer, renal-cellcarcinoma, liver cancer, non-small cell carcinoma of the lung, cancer ofthe small intestine and cancer of the esophagus. In one embodiment, thecancer is a melanoma, e.g., an advanced stage melanoma. Metastaticlesions of the aforementioned cancers can also be treated or preventedusing the methods and compositions of the invention. Examples of othercancers that can be treated include bone cancer, pancreatic cancer, skincancer, cancer of the head or neck, cutaneous or intraocular malignantmelanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of theanal region, stomach cancer, testicular cancer, uterine cancer,carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the cervix, carcinoma of the vagina, carcinoma of thevulva, Hodgkin Disease, non-Hodgkin lymphoma, cancer of the esophagus,cancer of the small intestine, cancer of the endocrine system, cancer ofthe thyroid gland, cancer of the parathyroid gland, cancer of theadrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer ofthe penis, chronic or acute leukemias including acute myeloid leukemia,chronic myeloid leukemia, acute lymphoblastic leukemia, chroniclymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma,cancer of the bladder, cancer of the kidney or ureter, carcinoma of therenal pelvis, neoplasm of the central nervous system (CNS), primary CNSlymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma,pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cellcancer, T-cell lymphoma, environmentally induced cancers including thoseinduced by asbestos, and combinations of said cancers. Treatment ofmetastatic cancers, e.g., metastatic cancers that express PD-L1 (Iwai etal. (2005) Int. Immunol. 17:133-144) can be effected using the antibodymolecules described herein.

“Polynucleotide” as used herein includes but is not limited to DNA, RNA,cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA),shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (shortnucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, syntheticRNA, and/or tRNA.

The term “isolated” as used herein refers to molecules or biologicals orcellular materials being substantially free from other materials. In oneaspect, the term “isolated” refers to nucleic acid, such as DNA or RNA,or protein or polypeptide (e.g., an antibody or derivative thereof), orcell or cellular organelle, or tissue or organ, separated from otherDNAs or RNAs, or proteins or polypeptides, or cells or cellularorganelles, or tissues or organs, respectively, that are present in thenatural source. The term “isolated” also refers to a nucleic acid orpeptide that is substantially free of cellular material, viral material,or culture medium when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized.Moreover, an “isolated nucleic acid” is meant to include nucleic acidfragments which are not naturally occurring as fragments and would notbe found in the natural state. The term “isolated” is also used hereinto refer to polypeptides which are isolated from other cellular proteinsand is meant to encompass both purified and recombinant polypeptides.The term “isolated” is also used herein to refer to cells or tissuesthat are isolated from other cells or tissues and is meant to encompassboth, cultured and engineered cells or tissues.

“Naked DNA” as used herein refers to DNA encoding a CAR cloned in asuitable expression vector in proper orientation for expression. Viralvectors which may be used include but are not limited SIN lentiviralvectors, retroviral vectors, foamy virus vectors, adeno-associated virus(AAV) vectors, hybrid vectors and/or plasmid transposons (for examplesleeping beauty transposon system) or integrase based vector systems.Other vectors that may be used in connection with alternate embodimentsof the invention will be apparent to those of skill in the art.

“Target cell” as used herein refers to cells which are involved in adisease and can be targeted by the genetically modified cells of theinvention (including but not limited to genetically modified T-cells, NKcells, hematopoietic stem cells, pluripotent stem cells, and embryonicstem cells). Other target cells will be apparent to those of skill inthe art and may be used in connection with alternate embodiments of theinvention.

“Vector”, “cloning vector” and “expression vector” as used herein referto the vehicle by which a polynucleotide sequence (e.g. a foreign gene)can be introduced into a host cell, so as to transform the host andpromote expression (e.g. transcription and translation) of theintroduced sequence. Vectors include plasmids, phages, viruses, etc.

As used herein, the term “administering,” refers to the placement anagent as disclosed herein into a subject by a method or route whichresults in at least partial localization of the agents at a desiredsite.

“Beneficial results” may include, but are in no way limited to,lessening or alleviating the severity of the disease condition,preventing the disease condition from worsening, curing the diseasecondition, preventing the disease condition from developing, loweringthe chances of a patient developing the disease condition and prolonginga patient's life or life expectancy. As non-limiting examples,“beneficial results” or “desired results” may be alleviation of one ormore symptom(s), diminishment of extent of the deficit, stabilized(i.e., not worsening) state of cancer progression, delay or slowing ofmetastasis or invasiveness, and amelioration or palliation of symptomsassociated with the cancer.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with, a disease ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorder, suchas cancer. Treatment is generally “effective” if one or more symptoms orclinical markers are reduced. Alternatively, treatment is “effective” ifthe progression of a disease is reduced or halted. That is, “treatment”includes not just the improvement of symptoms or markers, but also acessation of at least slowing of progress or worsening of symptoms thatwould be expected in absence of treatment. Beneficial or desiredclinical results include, but are not limited to, alleviation of one ormore symptom(s), diminishment of extent of disease, stabilized (i.e.,not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. The term “treatment” of a disease also includes providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment). In some embodiments, treatment of cancer includesdecreasing tumor volume, decreasing the number of cancer cells,inhibiting cancer metastases, increasing life expectancy, decreasingcancer cell proliferation, decreasing cancer cell survival, oramelioration of various physiological symptoms associated with thecancerous condition.

“Conditions” and “disease conditions,” as used herein may include,cancers, tumors or infectious diseases. In exemplary embodiments, theconditions include but are in no way limited to any form of malignantneoplastic cell proliferative disorders or diseases. In exemplaryembodiments, conditions include any one or more of kidney cancer,melanoma, prostate cancer, breast cancer, glioblastoma, lung cancer,colon cancer, or bladder cancer.

The term “effective amount” or “therapeutically effective amount” asused herein refers to the amount of a pharmaceutical compositioncomprising one or more peptides as disclosed herein or a mutant,variant, analog or derivative thereof, to decrease at least one or moresymptom of the disease or disorder, and relates to a sufficient amountof pharmacological composition to provide the desired effect. The phrase“therapeutically effective amount” as used herein means a sufficientamount of the composition to treat a disorder, at a reasonablebenefit/risk ratio applicable to any medical treatment.

A therapeutically or prophylactically significant reduction in a symptomis, e.g. at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 100%, atleast about 125%, at least about 150% or more in a measured parameter ascompared to a control or non-treated subject or the state of the subjectprior to administering the oligopeptides described herein. Measured ormeasurable parameters include clinically detectable markers of disease,for example, elevated or depressed levels of a biological marker, aswell as parameters related to a clinically accepted scale of symptoms ormarkers for diabetes. It will be understood, however, that the totaldaily usage of the compositions and formulations as disclosed hereinwill be decided by the attending physician within the scope of soundmedical judgment. The exact amount required will vary depending onfactors such as the type of disease being treated, gender, age, andweight of the subject.

The phrase “first line” or “second line” or “third line” refers to theorder of treatment received by a patient. First line therapy regimensare treatments given first, whereas second or third line therapy aregiven after the first line therapy or after the second line therapy,respectively. The National Cancer Institute defines first line therapyas “the first treatment given for a disease”, which is often part of astandard set of treatments. When used by itself, first-line therapy isthe one accepted as the best treatment. If it doesn't cure the diseaseor it causes severe side effects, other treatment may be added or usedinstead. It is also called induction therapy, primary therapy, andprimary treatment. See National Cancer Institute website, last visitedon Jun. 8, 2018. Typically, a patient is given a subsequent chemotherapyregimen because the patient did not show a positive clinical orsub-clinical response to the first line therapy or the first linetherapy has stopped.

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be included within the scope of this term.

“Particle” as used herein refers to particulate matters of various sizesand any shape. The appropriate particle size can vary based on thematerials used to make the particle, the active agent or therapeuticagent carried therein, and the functional groups and chemistry involvedfor conjugation with an immune effector cell, as will be appreciated bya person of skill in the art in light of the teachings disclosed herein.For example, the particles can be nanoparticles having an averageddiameter between 1 nm and 1,000 nm, or microparticles having an averageddiameter greater than 1 μm but about at least an order of magnitudesmaller than the immune effector cell to which the particles conjugated.For example, in some embodiments the particle has a diameter of fromabout 1 nm to about 1000 nm; or from about 25 nm to about 750 nm; orfrom about 50 nm to about 500 nm; or from about 100 nm to about 300 nm.In some embodiments, the average particle size can be about 1 nm, about10 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, or about1000 nm, or about 2,000 nm, or about 5,000 nm, or about 6,000 nm, orabout 10,000 nm. In some embodiments, the particle can be a nanoparticleor a microparticle, as these terms are defined herein. The particles canbe all nanoparticles, all microparticles, or a combination ofnanoparticles and microparticles. In some embodiments, the particles areliposomes. In other embodiments, the particles are polymeric particlesformed from biocompatible and/or biodegradable polymers. In someembodiments, the particles contain a core. In some embodiments, theparticles contain a coating.

“Biodegradable polymer” as used herein can contain a synthetic polymer,although natural polymers also can be used. The polymer can be, forexample, poly(lactic-co-glycolic acid) (PLGA), polystyrene orcombinations thereof. The polystyrene can, for example, be modified withcarboxy groups. Other examples of biodegradable polymers includepoly(hydroxy acid); poly(lactic acid); poly(glycolic acid); poly(lacticacid-co-glycolic acid); poly(lactide); poly(glycolide);poly(lactide-co-glycolide); polyanhydrides; polyorthoesters; polyamides;polycarbonates; polyalkylenes; polyethylene; polypropylene; polyalkyleneglycols; poly(ethylene glycol); polyalkylene oxides; poly(ethyleneoxides); polyalkylene terephthalates; poly(ethylene terephthalate);polyvinyl alcohols; polyvinyl ethers; polyvinyl esters; polyvinylhalides; poly(vinyl chloride); polyvinylpyrrolidone; polysiloxanes;poly(vinyl alcohols); poly(vinyl acetate); polyurethanes; co-polymers ofpolyurethanes; derivativized celluloses; alkyl cellulose; hydroxyalkylcelluloses; cellulose ethers; cellulose esters; nitro celluloses; methylcellulose; ethyl cellulose; hydroxypropyl cellulose; hydroxy-propylmethyl cellulose; hydroxybutyl methyl cellulose; cellulose acetate;cellulose propionate; cellulose acetate butyrate; cellulose acetatephthalate; carboxylethyl cellulose; cellulose triacetate; cellulosesulfate sodium salt; polymers of acrylic acid; methacrylic acid;copolymers of methacrylic acid; derivatives of methacrylic acid;poly(methyl methacrylate); poly(ethyl methacrylate);poly(butylmethacrylate); poly(isobutyl methacrylate);poly(hexylmethacrylate); poly(isodecyl methacrylate); poly(laurylmethacrylate); poly(phenyl methacrylate); poly(methyl acrylate);poly(isopropyl acrylate); poly(isobutyl acrylate); poly(octadecylacrylate); poly(butyric acid); poly(valeric acid);poly(lactide-co-caprolactone); copolymers ofpoly(lactide-co-caprolactone); blends of poly(lactide-co-caprolactone);poly-(isobutyl cyanoacrylate); poly(2-hydroxyethyl-L-glutamnine); andcombinations, copolymers and/or mixtures of one or more of any of theforegoing. Furthermore, as a person of ordinary skill in the art wouldappreciate, some of the polymers listed above as “biocompatible” canalso be considered biodegradable, whether or not they are included inthe above listing of representative biodegradable polymers. As usedherein, “derivatives” include polymers having substitutions, additionsof chemical groups and other modifications routinely made by thoseskilled in the art.

“Cytotoxicity” refers to an agent being toxic to cells, which may bequantified as the extent of cell death (e.g., number of dead cells as apercentage of the original cell number before incubation with the agent)over a period of incubation time with the cells. For example,cytotoxicity is quantified as the number of dead cells as a percentageof the original cell number before incubation with the agent over 24hours with the cells.

Without being bound to a particular theory, the efficacy of chemotherapyis enhanced as described in the present invention when tumor specificNatural Killer (NK) cells are used as carriers to deliver drug-loadednanoparticles. In some embodiments, tumor-specific NK cells containingchimeric antigen receptors (CAR.NK) and crosslinked multilamellarliposomal vesicles (cMLVs) that encapsulate paclitaxel (PTX). In variousaspects, these cMLVs are liposomes functionalized with thiol-reactivemaleimide headgroups, which allow them to be stably conjugated to thethiol-rich NK cell surface. This composition and/or delivery systemallows for combinatory drug delivery by co-localizing chemotherapeuticsand immune effector cells to a single site (close proximity), inducing asynergistic anti-tumor effect in vitro and in vivo. Described herein isthe combination of immunotherapy and chemotherapeutic drug delivery byutilizing CAR.NK cells as carriers for PTX-loaded crosslinkedmultilamellar liposomal vesicles (cMLV (PTX)) to enhance antitumorefficacy in Her2 and CD19 overexpressing cancer models (FIG. 1A).

Composition

In some embodiments of the inventions, provided herein are geneticallyengineered cells which include vectors that express antigen-specificchimeric antigen receptors (CARs) and further include drug-loadedparticles bound to the cell surface. In some embodiments, the particlesare liposomes (e.g., crosslinked multilamellar vesicles) which areloaded with chemotherapeutic agents. In some embodiments, the CARtargets one antigen. In another embodiment, the CAR is a bispecific CARand targets two different antigens. The bispecific CARs may targetantigens on the same type of target cells or different cells.

In various embodiments, the genetically engineered cells expressingantigen-specific CARs and surface conjugated with therapeutics-loadedliposomes are T cells or Natural Killer (NK) cells. In one embodiment,the genetically engineered cells expressing CARs and surface conjugatedwith therapeutics-loaded liposomes are genetically engineered NK cells.

In various embodiments, the liposomes are multilamellar vesicle (withseveral lamellar phase lipid bilayers), small unilamellar liposomevesicle (with one lipid bilayer), large unilamellar vesicle or cochleatevesicle.

In some embodiments, the antigens which may be targeted by the CARs whenexpressed in cells (such as NK cells) as described herein include butare not limited to any one or more of CD19, CD22, CD23, MPL, CD123,CD32, CD138, CD200R, CD276, CD324, CD30, CD32, FcRH5, CD99, TissueFactor, amyloid, Fc region of an immunoglobulin, CD171, CS-1, CLL-1(CLECL1), CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP,TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL11Ra,Mesothelin, PSCA, VEGFR2, Lewis Y, CD24, PDGFR-beta, PRSS21, SSEA-4,CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM,Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100,bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLea, GM3, TGS5, BMWMAA,o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR,TCR-beta1 constant chain, TCR beta2 constant chain, TCR gamma-delta,GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH,NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1,NY-ESO-1, LAGE-1a, legumain, HPV E6, E7, HTLV1-Tax, KSHV K8.1 protein,EBB gp350, HIV1-envelop glycoprotein gp120, MAGE-A1, MAGE A1, ETV6-AML,sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1/Galectin8, MelanA/MART1, Ras mutant, hTERT, DLL3, TROP2, PTK7, GCC, AFP, sarcomatranslocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17,PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS,SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, RU1, RU2, intestinalcarboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2,CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, FITC,Leutenizing hormone receptor (LHR), Follicle stimulating hormonereceptor (FSHR), Chorionic Gonadotropin Hormone receptor (CGHR), CCR4,GD3, SLAMF6, SLAMF4, FITC, Leutenizing hormone receptor (LHR), Folliclestimulating hormone receptor (FSHR), Chorionic Gonadotropin Hormonereceptor (CGHR), CCR4, GD3, SLAMF6, SLAMF4, or combinations thereof.

In various embodiments, chemotherapeutic agents that may be encapsulatedor otherwise delivered with the liposomes or polymeric particles includebut are not limited to any one or more of Temozolomide, Actinomycin,Alitretinoin, All-trans retinoic acid, Azacitidine, Azathioprine,Bevacizumab, Bexatotene, Bleomycin, Bortezomib, Carboplatin,Capecitabine, Cetuximab, Cisplatin, Chlorambucil, Cyclophosphamide,Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin,liposome-encapsulated Doxorubicin such as as Doxil (pegylated form),Myocet (nonpegylated form) and Caelyx, Epirubicin, Epothilone,Erlotinib, Etoposide, Fluorouracil, Folinic acid, Gefitinib,Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Ipilimumab, Irinotecan,Nanoliposomal Irinotecan (Nal-IRI), Mechlorethamine, Melphalan,Mercaptopurine, Methotrexate, Mitoxantrone, Ocrelizumab, Ofatumumab,Oxaliplatin, Paclitaxel, Taxol, Abraxane, Genexol, Protein-BoundPaclitaxel, Nab-Paclitaxel, Panitumab, Pemetrexed, Rituximab,Tafluposide, Teniposide, Tioguanine, Topotecan, Tretinoin, Valrubicin,Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine,Vorinostat, Romidepsin, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP),Cladribine, Clofarabine, Floxuridine, Fludarabine, Pentostatin,Mitomycin, ixabepilone, Estramustine, prednisone, methylprednisolone,dexamethasone or a combination thereof.

In various embodiments, the chemotherapeutic agent is a platinum-basedantineoplastic agent. Examples of the platinum-based antineoplasticagent include but are not limited to oxaliplatin, cisplatin, lipoplatin(a liposomal version of cisplatin), carboplatin, satraplatin,picoplatin, nedaplatin, and triplatin, and their functional equivalents,analogs, derivatives, variants or salts.

Generally, particles are conjugated to each cell at a ratio that doesnot negatively alter the function of the cell, yet high enough todeliver a high load of active agent per cell. For example, the number ofconjugated nanoparticles (e.g., cMLVs) per cell is between 150 and 100,between 200 and 150, between 250 and 200, between 300 and 250, between350 and 300, or between 400 and 350.

In some embodiments, the active agent, e.g., chemotherapeutics, inparticles are delivered in an amount that does not cause cytotoxicity tothe engineered NK cells following administration, yet high enough toinhibit or kill tumor cells in vitro and in vivo. In other embodiments,the active agent such as chemotherapeutics are carried in particles onCAR-expressing immune effector cells in an amount that causescytotoxicity to less than 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% of normalcells, yet high enough to inhibit or kill more than 10%, 15%, or 20% oftumor cells. In yet another embodiment, the active agent such aschemotherapeutics are carried in particles on CAR-expressing immuneeffector cells in an amount that causes cytotoxicity (e.g., death) toless than 5%, 6%, 7%, 8%, 9%, or 10% of a population of engineered NKcells, yet high enough to inhibit or kill more than 10%, 15%, or 20% oftumor cells.

In some embodiments, compositions are provided including geneticallyengineered NK cells, wherein the NK cells express one or more CARs andhaving bound on the surface crosslinked multilamellar liposomal vesicles(cMLVs) which encapsulate chemotherapeutic agents. In some embodiments,compositions are provided which includes genetically engineered NKcells, wherein the NK cells express CARs that target Her2 and arechemically bonded on the surface with a plurality of cMLVs thatencapsulate chemotherapeutic agents. In some embodiments, providedherein are compositions including genetically engineered NK cells,wherein the NK cells express CARs that target CD19 and are chemicallybonded on the surface with a plurality of cMLVs that encapsulatechemotherapeutic agents. In some embodiments, provided herein arecompositions including genetically engineered NK cells, wherein the NKcells express CARs that target CD19 and Her2 and are chemically bondedon the surface with a plurality of cMLVs that encapsulatechemotherapeutic agents.

In some embodiments, provided herein are compositions includinggenetically engineered NK cells, wherein the NK cells express one ormore CARs and are chemically bonded on the surface with a plurality ofcMLVs that encapsulate paclitaxel. In some embodiments, provided hereinare compositions including genetically engineered NK cells, wherein theNK cells express CARs that target Her2 and are chemically bonded on thesurface with a plurality of cMLVs that encapsulate paclitaxel. In someembodiments, provided herein are compositions including geneticallyengineered NK cells, wherein the NK cells express CARs that target CD19and are chemically bonded on the surface with a plurality of cMLVs thatencapsulate paclitaxel. In some embodiments, provided herein arecompositions including genetically engineered NK cells, wherein the NKcells express CARs that target CD19 and Her2 and are chemically bondedon the surface with a plurality of cMLVs that encapsulate paclitaxel.

Various embodiments provide crosslinked multilamellar liposomes as theactive agent carrier, which are bound to the surface of an immuneeffector cell. A crosslinked multilamellar liposome has an exteriorsurface and an interior surface, the interior surface defining a centralliposomal cavity. The multilamellar liposome includes at least a firstlipid bilayer and a second lipid bilayer, the first lipid bilayer beingcovalently bonded to the second lipid bilayer. In one aspect, the lipidbilayers are covalently bonded by ether bonds and/or thioether bonds.Typically, multilamellar liposome includes at least one additional lipidbilayer such as third lipid bilayer which is covalently bonded to secondlipid bilayer. In one embodiment, multilamellar liposome includes onaverage from 2 to 10 lipid bilayers. In another embodiment,multilamellar liposome includes on average from 3 to 9 lipid bilayers.In still another embodiment, multilamellar liposome includes on averagefrom 3 to 6 lipid bilayers. In some variations, poly(alkylene glycol)groups (e.g., poly(ethylene glycol)) are covalently bonded to theexterior surface of the liposome in order to improve water solubility.For example, the poly(ethylene glycol) groups have a weight averagemolecular weight from about 400 to 2500 Daltons. In another refinementthe poly(ethylene glycol) groups include from 9 to 45 repeat units of—OCH₂CH₂—.

Various chemical and/or physical interactions can be employed to bind aplurality of nanoparticles (e.g., cMLVs) to the surface of immuneeffector cells. In various embodiments, active agent-carryingnanoparticles are chemically bonded to the surface of the cell. In oneembodiment, maleimide group is functionalized on the nanoparticles,which can chemically bond with the free thiols on the immune effectorcells. Optionally, a linker between the nanoparticles and the cellsurface is present, e.g., via a polyethylene glycol. Various embodimentsprovide that bound cMLVs on the surface of NK cells are not internalizedor phagocytized by the NK cells.

Various embodiments provide at least one active agent, e.g., anticancercompound, is carried by a multilamellar liposome, through physicalencapsulation, entrapment or chemical bonding. In one embodiment, anactive agent can be disposed within the cavity of a crosslinkedmultilamellar liposome. In another embodiment, an active agent isdisposed within the lipid bilayers and any additional lipid layers.

Therapeutic Methods

Provided herein are methods for treating, inhibiting, preventingmetastasis of and/or reducing severity of cancer in a subject in needthereof. The methods include administering to the subject an effectiveamount of a composition described herein.

In some embodiments, provided herein are methods for treating,inhibiting, preventing metastasis of and/or reducing severity of cancerin a subject in need thereof by administering to the subject aneffective amount of a composition comprising genetically engineered NKcells which express CARs and are chemically bonded on the surface with aplurality of particles that encapsulate chemotherapeutic agents.

In some embodiments of the therapeutic methods described herein, theantigens which may be targeted by the CARs when expressed in cells (suchas NK cells) as described herein include but are not limited to any oneor more of CD19, CD22, CD23, MPL, CD123, CD32, CD138, CD200R, CD276,CD324, CD30, CD32, FcRH5, CD99, Tissue Factor, amyloid, Fc region of animmunoglobulin, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII, GD2, GD3,BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM,B7H3, KIT, IL-13Ra2, IL11Ra, Mesothelin, PSCA, VEGFR2, Lewis Y, CD24,PDGFR-beta, PRSS21, SSEA-4, CD20, Folate receptor alpha, ERBB2(Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-Ireceptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1,sLea, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248,TEM7R, CLDN6, TSHR, TCR-beta1 constant chain, TCR beta2 constant chain,TCR gamma-delta, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid,PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2,TARP, WT1, NY-ESO-1, LAGE-1a, legumain, HPV E6, E7, HTLV1-Tax, KSHV K8.1protein, EBB gp350, HIV1-envelop glycoprotein gp120, MAGE-A1, MAGE A1,ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2,Fos-related antigen 1, p53, p53 mutant, prostein, survivin andtelomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, DLL3,TROP2, PTK7, GCC, AFP, sarcoma translocation breakpoints, ML-IAP, ERG(TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1,MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4,SSX2, RAGE-1, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2,CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2,LY75, GPC3, FCRL5, IGLL1, FITC, Leutenizing hormone receptor (LHR),Follicle stimulating hormone receptor (FSHR), Chorionic GonadotropinHormone receptor (CGHR), CCR4, GD3, SLAMF6, SLAMF4, FITC, Leutenizinghormone receptor (LHR), Follicle stimulating hormone receptor (FSHR),Chorionic Gonadotropin Hormone receptor (CGHR), CCR4, GD3, SLAMF6,SLAMF4, or combinations thereof.

In various embodiments of the therapeutic methods described herein,chemotherapeutic agents optionally in combination with other classes ofcompounds that may be encapsulated or otherwise delivered (e.g.,including chemically bonded) with multilamellar liposomal vesiclesinclude but are not limited to any one or more of Temozolomide,Actinomycin, Alitretinoin, All-trans retinoic acid, Azacitidine,Azathioprine, Bevacizumab, Bexatotene, Bleomycin, Bortezomib,Carboplatin, Capecitabine, Cetuximab, Cisplatin, Chlorambucil,Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine,Doxorubicin, liposome-encapsulated Doxorubicin such as Doxil (pegylatedform), Myocet (nonpegylated form) and Caelyx, Epirubicin, Epothilone,Erlotinib, Etoposide, Fluorouracil, Folinic acid, Gefitinib,Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Ipilimumab, Irinotecan,Nanoliposomal Irinotecan (Nal-IRI), Mechlorethamine, Melphalan,Mercaptopurine, Methotrexate, Mitoxantrone, Ocrelizumab, Ofatumumab,Oxaliplatin, Paclitaxel, Taxol, Abraxane, Genexol, Protein-BoundPaclitaxel, Nab-Paclitaxel, Panitumab, Pemetrexed, Rituximab,Tafluposide, Teniposide, Tioguanine, Topotecan, Tretinoin, Valrubicin,Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine,Vorinostat, Romidepsin, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP),Cladribine, Clofarabine, Floxuridine, Fludarabine, Pentostatin,Mitomycin, ixabepilone, Estramustine, prednisone, methylprednisolone,dexamethasone or a combination thereof.

In one embodiment, the cancer specific antigen is expressed on bothnormal cells and cancers cells, but is expressed at lower levels onnormal cells. In one embodiment, the method further comprises selectinga CAR that binds the cancer specific antigen of interest with anaffinity that allows the antigen specific CAR to bind and kill thecancer cells. In some embodiments, the antigen specific CAR kills cancercells but kills less than 30%, 25%, 20%, 15%, 10%, 5% or less of thenormal cells expressing the cancer antigen. In exemplary embodiments,the percentage of cells killed by the antigen specific CARs may bedetermined using the cell death assays described herein.

In some embodiments, provided herein are methods for treating,inhibiting, preventing metastasis of and/or reducing severity of cancerin a subject in need thereof by administering to the subject aneffective amount of a composition comprising NK cells that express CARsthat target CD19 and the cells being chemically bonded on the surfacewith a plurality of cMLVs that encapsulate chemotherapeutic agents(e.g., paclitaxel).

In some embodiments, provided herein are methods for treating,inhibiting, preventing metastasis of and/or reducing severity of cancerin a subject in need thereof by administering to the subject aneffective amount of a composition comprising NK cells that express CARsthat target Her2 and the cells being chemically bonded on the surfacewith a plurality of cMLVs that encapsulate chemotherapeutic agents(e.g., paclitaxel).

In some embodiments, provided herein are methods for treating,inhibiting, preventing metastasis of and/or reducing severity of cancerin a subject in need thereof comprising administering to the subject aneffective amount of a composition comprising NK cells that express CARsthat target CD19 and Her2 and the cells being chemically bonded on thesurface with a plurality of cMLVs that encapsulate chemotherapeuticagents (e.g., paclitaxel).

Exemplary cancers whose growth can be inhibited include cancerstypically responsive to immunotherapy. Non-limiting examples of cancersfor treatment include melanoma (e.g., metastatic malignant melanoma),renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormonerefractory prostate adenocarcinoma), breast cancer, colon cancer andlung cancer (e.g. non-small cell lung cancer). Additionally, refractoryor recurrent malignancies can be treated using the compositionsdescribed herein. In one embodiment, the engineered immune effector celldescribed herein is used for treatment of a subject with ovarian tumor.In another embodiment, the engineered immune effector cell describedherein is used for treatment of a subject with melanoma. In yet anotherembodiment, the engineered immune effector cell described herein is usedfor treatment of a subject with renal cancer. Another embodimentprovides the engineered immune effector cell described herein is usedfor treatment of a subject with prostate cancer. The engineered immuneeffector cell described herein can also be used for treatment of asubject with breast cancer, lung cancer, or both. Another embodimentprovides the engineered immune effector cell described herein is usedfor treatment of a subject with leukemia.

In exemplary embodiments, cancers treated by the methods describedherein include solid tumors such as sarcomas, adenocarcinomas, andcarcinomas, of the various organ systems, such as those affecting liver,lung, breast, lymphoid, gastrointestinal (e.g., colon), genitourinarytract (e.g., renal, urothelial cells), prostate and pharynx.Adenocarcinomas include malignancies such as most colon cancers, rectalcancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma ofthe lung, cancer of the small intestine and cancer of the esophagus. Inone embodiment, the cancer is a melanoma, e.g., an advanced stagemelanoma. Metastatic lesions of the aforementioned cancers can also betreated or prevented using the methods and compositions of theinvention.

Examples of other cancers that can be treated include bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular malignant melanoma, uterine cancer, ovarian cancer, rectalcancer, cancer of the anal region, stomach cancer, testicular cancer,uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina, carcinomaof the vulva, Hodgkin Disease, non-Hodgkin lymphoma, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, chronic or acute leukemias including acutemyeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers. Treatment of metastatic cancers, e.g.,metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol.17:133-144) can be effected using the antibody molecules describedherein. Further a disease associated with a cancer associate antigen asdescribed herein expression include, but not limited to, e.g., atypicaland/or non-classical cancers, malignancies, precancerous conditions orproliferative diseases associated with expression of a cancer associateantigen as described herein. In some embodiments, a CAR-expressing Tcell or NK cell as described herein reduces the quantity, number, amountor percentage of cells and/or cancer cells by at least 25%, at least30%, at least 40%, at least 50%, at least 65%, at least 75%, at least85%, at least 95%, or at least 99% in a subject with hematologicalcancer or another cancer associated with a cancer associated antigen asdescribed herein, expressing cells relative to a negative control. Inone embodiment, the subject is a human.

In some embodiments, the therapeutically effective amount of thegenetically modified cells as described herein (for example, NK cellsexpressing CARs and further chemically bonded on the surface with aplurality of particles that encapsulate chemotherapeutic agents) isadministered at a dosage of 10⁴ to 10⁹ cells/kg body weight, in someinstances 10⁵ to 10⁶ cells/kg body weight, including all integer valueswithin those ranges. In other instances, between about 0.1×10⁹ and0.5×10⁹, between about 0.5×10⁹ and 1.0×10⁹, or between about 1.0×10⁹ and5.0×10⁹ engineered immune effector cells are administered per injectionto a human subject. Various embodiments provide that the immune effectorcells are administered one or more times. Subsequent administrationstypically occur at weekly, biweekly, triweekly, monthly, quarterly oryearly intervals, or at a combination of the frequencies mentionedabove. T cell compositions may also be administered multiple times atthese dosages. The cells can be administered by using infusiontechniques that are commonly known in immunotherapy (see, e.g.,Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The cells can beadministered by injection into the site of the lesion (e.g.,intra-tumoral injection).

In some embodiments, the therapeutic methods described herein furtherincludes administering to the subject, sequentially or simultaneously,existing therapies. Examples of existing cancer treatment include, butare not limited to, active surveillance, observation, surgicalintervention, chemotherapy, immunotherapy, radiation therapy (such asexternal beam radiation, stereotactic radiosurgery (gamma knife), andfractionated stereotactic radiotherapy (FSR)), focal therapy, systemictherapy, vaccine therapies, viral therapies, molecular targetedtherapies, or combinations thereof.

Also provided herein are methods for preparing genetically engineeredcells comprising transfecting the cells with vectors comprising nucleicacid encoding the CARs described herein. In some embodiments, the cellsare immune effector cells, such as human T cells or human NK cells, orstem cells that give rise to immune effector cells. In some embodiments,the cells are autologous human T cells or autologous human NK cells orautologous human stem cells. In some embodiments, the cells areallogeneic human T cells or allogeneic human NK cells or allogeneichuman stem cells.

In some embodiments, methods for preparing the genetically modifiedcells comprise obtaining a population of cells and selecting cells thatexpress any one or more of CD3, CD28, CD4, CD8, CD45RA, and/or CD45RO.In certain embodiments, the population of immune effector cells providedare CD3+ and/or CD28+.

In one embodiment, the method for preparing the genetically modifiedcells comprise obtaining a population of cells and enriching for theCD25+T regulatory cells, for example by using antibodies specific toCD25. Methods for enriching CD25+T regulatory cells from the populationof cells will be apparent to a person of skill in the art. In someembodiments, the Treg enriched cells comprise less than 30%, 20%, 10%,5% or less non-Treg cells. In some embodiments, the vectors encoding theCARs described herein are transfected into Treg-enriched cells. Tregenriched cells expressing a CAR may be used to induced tolerance toantigen targeted by the CAR.

In some embodiments, the method further comprises expanding thepopulation of cells after the vectors comprising nucleic acids encodingthe CARs described herein have been transfected into the cells. Inembodiments, the population of cells is expanded for a period of 8 daysor less. In certain embodiments, the population of cells is expanded inculture for 5 days, and the resulting cells are more potent than thesame cells expanded in culture for 9 days under the same cultureconditions. In other embodiments, the population of cells is expanded inculture for 5 days show at least a one, two, three or four fold increasein cell doublings upon antigen stimulation as compared to the same cellsexpanded in culture for 9 days under the same culture conditions. Insome embodiments, the population of cells is expanded in an appropriatemedia that includes one or more interleukins that result in at least a200-fold, 250-fold, 300-fold, or 350-fold increase in cells over a 14day expansion period, as measured by flow cytometry.

In various embodiments, the expanded cells comprise one or more CARs andfurther comprise liposomes (for example, multilamellar liposomalvesicles) conjugated to chemotherapeutic agents, as described herein. Insome embodiments, the expanded cells comprise one CAR with one, two,three or more ASDs. In some embodiments, the expanded cells furthercomprise accessory modules and therapeutic controls as described herein.

Combination Therapies

Therapeutic methods described herein include using compositions thathave genetically modified cells which contain nucleic acids encodingCARs and are surface bonded with a plurality of chemotherapeuticagents-loaded particles. In various embodiments, the therapeutic methodsdescribed herein may be combined with existing therapies and agents. Thetherapeutic compositions described herein, e.g., genetically modifiedcells which contain nucleic acids encoding CARs and are surface bondedwith a plurality of chemotherapeutic agents-loaded particles, areadministered to the subject with at least one additional known therapyor therapeutic agent. In some embodiments, the compositions describedherein and the additional therapy or therapeutic agents are administeredsequentially. In some embodiments, the compositions described herein andthe additional therapy or therapeutic agents are administeredsimultaneously. The optimum order of administering the compositionsdescribed herein and the existing therapies will be apparent to a personof skill in the art, such as a physician.

A genetically engineered CAR-expressing cell further including on thesurface a plurality of chemotherapeutic agent-loaded nanoparticles, asdescribed herein and the at least one additional therapeutic agent canbe administered simultaneously, in the same or in separate compositions,or sequentially. For sequential administration, the cells describedherein can be administered first, and the additional agent can beadministered second, or the order of administration can be reversed.

Combinations therapies may be administered to the subject over theduration of the disease. Duration of the disease includes from diagnosisuntil conclusion of treatment, wherein the treatment results inreduction of symptoms and/or elimination of symptoms. In variousembodiments, the effect of the two treatments can be partially additive,wholly additive, or greater than additive. The delivery can be such thatan effect of the first treatment delivered is still detectable when thesecond is delivered.

Therapy using the cells described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded nanoparticles) and/or other therapeuticagents, procedures or modalities can be administered during periods ofactive disorder, or during a period of remission or less active disease.Therapy using the cells described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) can be administered before theother treatment, concurrently with the treatment, post-treatment, orduring remission of the disorder.

When administered in combination, the therapy using the cells describedherein (for example, NK cells expressing CARs and further including onthe surface a plurality of chemotherapeutic agent-loaded cMLVs) and theadditional agent (e.g., second or third agent), or all, can beadministered in an amount or dose that is higher, lower or the same thanthe amount or dosage of each agent used individually, e.g., as amonotherapy. In certain embodiments, the administered amount or dosageof the therapy using the cells described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs), the additional agent (e.g., secondor third agent), or all, is lower (e.g., at least 20%, at least 30%, atleast 40%, or at least 50%) than the amount or dosage of each agent usedindividually, e.g., as a monotherapy. In other embodiments, the amountor dosage of the therapy using the cells described herein (for example,NK cells expressing CARs and further including on the surface aplurality of chemotherapeutic agent-loaded cMLVs), the additional agent(e.g., second or third agent), or all, that results in a desired effect(e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%,at least 40%, or at least 50% lower) than the amount or dosage of eachagent used individually, e.g., as a monotherapy, required to achieve thesame therapeutic effect.

Further method aspects relate administering to the subject an effectiveamount of the cell described herein (for example, NK cells expressingCARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs), optionally in combination with anagent that increases the efficacy and/or safety of the immune cell. Infurther aspects, the agent that increases the efficacy and/or safety ofthe immune cell is one or more of: (i) a protein phosphatase inhibitor;(ii) a kinase inhibitor; (iii) a cytokine; (iv) an inhibitor of animmune inhibitory molecule; or (v) an agent that decreases the level oractivity of a TREG cell; vi) an agent that increase the proliferationand/or persistence of CAR-modified cells vii) a chemokine viii) an agentthat increases the expression of CAR ix) an agent that allows regulationof the expression or activity of CAR x) an agent that allows controlover the survival and/or persistence of CAR-modified cells xi) an agentthat controls the side effects of CAR-modified cells xii) a Brd4inhibitor xiii) an agent that delivers a therapeutic (e.g. sHVEM) orprophylactic agent to the site of the disease xiv) an agent thatincreases the expression of the target antigen against which CAR isdirected; xv) an adenosine A2a receptor antagonist

In some embodiments, the genetically modified cells described herein(for example, NK cells expressing CARs and further including on thesurface a plurality of chemotherapeutic agent-loaded cMLVs) may be usedin a treatment regimen in combination with surgery, chemotherapy,radiation, immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin,mycophenolic acid, steroids, FR901228, cytokines, and irradiation,peptide vaccine, such as that described in Izumoto et al. 2008 JNeurosurg 108:963-971. In one embodiment, a CAR-expressing celldescribed herein can be used in combination with a chemotherapeuticagent. Exemplary chemotherapeutic agents include an anthracycline (e.g.,doxorubicin (e.g., liposomal doxorubicin)), a vinca alkaloid (e.g.,vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent(e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide,temozolomide), an immune cell antibody (e.g., alemtuzamab, gemtuzumab,rituximab, ofatumumab, tositumomab, brentuximab), an antimetabolite(including, e.g., folic acid antagonists, pyrimidine analogs, purineanalogs and adenosine deaminase inhibitors (e.g., fludarabine)), an mTORinhibitor, a TNFR glucocorticoid induced TNFR related protein (GITR)agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin orbortezomib), an immunomodulator such as thalidomide or a thalidomidederivative (e.g., lenalidomide).

In embodiments, the cell described herein (for example, NK cellsexpressing CARs and including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with cyclophosphamide and fludarabine.

In embodiments, the cell described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with bendamustine and rituximab. In embodiments, the subjecthas CLL.

In embodiments, the cell described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with rituximab, cyclophosphamide, doxorubicin, vincristine,and/or a corticosteroid (e.g., prednisone). In embodiments, aCAR-expressing cell described herein is administered to a subject incombination with rituximab, cyclophosphamide, doxorubicin, vincristine,and prednisone (R-CHOP). In embodiments, the subject has diffuse largeB-cell lymphoma (DLBCL).

In embodiments, the cell described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with etoposide, prednisone, vincristine, cyclophosphamide,doxorubicin, and/or rituximab. In embodiments, a CAR-expressing celldescribed herein is administered to a subject in combination withetoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, andrituximab (EPOCH-R). In embodiments, a CAR-expressing cell describedherein is administered to a subject in combination with dose adjustedEPOCH-R (DA-EPOCH-R). In embodiments, the subject has a B cell lymphoma,e.g., a Myc-rearranged aggressive B cell lymphoma.

In embodiments, the described herein (for example, NK cells expressingCARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with brentuximab. Brentuximab is an antibody-drug conjugateof anti-CD30 antibody and monomethyl auristatin E. In embodiments, thesubject has Hodgkin's lymphoma (HL), e.g., relapsed or refractory HL. Inembodiments, the subject comprises CD30+HL. In embodiments, the subjecthas undergone an autologous stem cell transplant (ASCT).

In some embodiments, the cell described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with a CD20 inhibitor, e.g., an anti-CD20 antibody (e.g., ananti-CD20 mono- or bispecific antibody) or a fragment thereof.

In one embodiment, the described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with an mTOR inhibitor, e.g., an mTOR inhibitor describedherein, e.g., a rapalog such as everolimus. In one embodiment, the mTORinhibitor is administered prior to the CAR-expressing cell. For example,in one embodiment, the mTOR inhibitor can be administered prior toapheresis of the cells. In one embodiment, the subject has CLL.

In one embodiment, the cell described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) can be used in combination with akinase inhibitor. In one embodiment, the kinase inhibitor is a CDK4inhibitor, e.g., a CDK4 inhibitor described herein, e.g., a CD4/6inhibitor, such as, e.g.,6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one,hydrochloride (also referred to as palbociclib or PD0332991). In oneembodiment, the kinase inhibitor is a BTK inhibitor, e.g., a BTKinhibitor described herein, such as, e.g., ibrutinib. In someembodiments, ibrutinib is administered at a dosage of about 300-600mg/day (e.g., about 300-350, 350-400, 400-450, 450-500, 500-550, or550-600 mg/day, e.g., about 420 mg/day or about 560 mg/day), e.g.,orally. In embodiments, the ibrutinib is administered at a dose of about250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg,520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g., 250 mg, 420 mg or 560 mg)daily for a period of time, e.g., daily for 21 day cycle, or daily for28 day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or more cycles of ibrutinib are administered. Without being bound bytheory, it is thought that the addition of ibrutinib enhances the T cellproliferative response and may shift T cells from a T-helper-2 (Th2) toT-helper-1 (Th1) phenotype. Th1 and Th2 are phenotypes of helper Tcells, with Th1 versus Th2 directing different immune response pathways.A Th1 phenotype is associated with proinflammatory responses, e.g., forkilling cells, such as intracellular pathogens/viruses or cancerouscells, or perpetuating autoimmune responses. A Th2 phenotype isassociated with eosinophil accumulation and anti-inflammatory responses.In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., anmTOR inhibitor described herein, such as, e.g., rapamycin, a rapamycinanalog, OSI-027. The mTOR inhibitor can be, e.g., an mTORC1 inhibitorand/or an mTORC2 inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2inhibitor described herein. In one embodiment, the kinase inhibitor is aMNK inhibitor, e.g., a MNK inhibitor described herein, such as, e.g.,4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d] pyrimidine. The MNKinhibitor can be, e.g., a MNK1a, MNK1b, MNK2a and/or MNK2b inhibitor. Inone embodiment, the kinase inhibitor is a dual PI3K/mTOR inhibitordescribed herein, such as, e.g., PF-04695102. In one embodiment, thekinase inhibitor is a Src kinase inhibitor. In one embodiment, thekinase inhibitor is Dasatinib. In one embodiment, the Src kinaseinhibitor is administered to the patient after the administration of CARexpressing cells to control or terminate the activity of CAR-expressingcells. In one embodiment, Dasatinib is administered to the patient afterthe administration of CAR expressing cells to control or terminate theactivity of CAR-expressing cells. In one embodiment, dasatinib isadministered orally at a dose of at least 10 mg/day, 20 mg/day, 40mg/day, 60 mg/day, 70 mg/day, 90 mg/day, 100 mg/day, 140 mg/day, 180mg/day or 210 mg/day.

In embodiments, the cell described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with an anaplastic lymphoma kinase (ALK) inhibitor.Exemplary ALK kinases include but are not limited to crizotinib(Pfizer), ceritinib (Novartis), alectinib (Chugai), brigatinib (alsocalled AP26113; Ariad), entrectinib (Ignyta), PF-06463922 (Pfizer),TSR-011 (Tesaro) (see, e.g., Clinical Trial Identifier No. NCT02048488),CEP-37440 (Teva), and X-396 (Xcovery). In some embodiments, the subjecthas a solid cancer, e.g., a solid cancer described herein, e.g., lungcancer.

Drugs that inhibit either the calcium dependent phosphatase calcineurin(cyclosporine and FK506) or inhibit the p70S6 kinase that is importantfor growth factor induced signaling (rapamycin) can also be used. In afurther aspect, the cell compositions of the present invention may beadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, and/orantibodies such as OKT3 or CAMP ATH. In one aspect, the cellcompositions of the present invention are administered following B-cellablative therapy such as agents that react with CD20, e.g., Rituxan. Forexample, in one embodiment, subjects may undergo standard treatment withhigh dose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentinvention. In an additional embodiment, expanded cells are administeredbefore or following surgery.

In embodiments, the cell described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with an autologous stem cell transplant, an allogeneic stemcell transplant, an autologous bone marrow transplant or an allogeneicbone marrow transplant.

In embodiments, the cell described herein (for example, NK cellsexpressing CARs and including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with microtransplant or HLA mismatched allogeneic cellulartherapy.

In embodiments, the cell described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with an indoleamine 2,3-dioxygenase (IDO) inhibitor.

In embodiments, the cell described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with a modulator of myeloid-derived suppressor cells(MDSCs). MDSCs accumulate in the periphery and at the tumor site of manysolid tumors. These cells suppress T cell responses, thereby hinderingthe efficacy of CAR-expressing cell therapy. Without being bound bytheory, it is thought that administration of a MDSC modulator enhancesthe efficacy of a CAR-expressing cell described herein. In anembodiment, the subject has a solid tumor, e.g., a solid tumor describedherein, e.g., glioblastoma. Exemplary modulators of MDSCs include butare not limited to MCS11O and BLZ945. MCS11O is a monoclonal antibody(mAb) against macrophage colony-stimulating factor (M-CSF). BLZ945 is asmall molecule inhibitor of colony stimulating factor 1 receptor(CSF1R). In embodiments, the cell described herein (for example, NKcells expressing CARs and further including on the surface a pluralityof chemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with a Brd4 or BET (bromodomain and extra-terminal motif)inhibitor. BET protein BRD4 directly regulated expression of thetranscription factor BATF in CD8+ T cells, which was associated withdifferentiation of T cells into an effector memory phenotype. JQ1, aninhibitor of bromodomain and extra-terminal motif (BET) proteins,maintained CD8+ T cells with functional properties of stem cell-like andcentral memory T cells. Exemplary Brd4 inhibitors that can beadministered in combination with CAR-expressing cells include but arenot limited to JQ1, MS417, OTXO15, LY 303511 and Brd4 inhibitor asdescribed in US 20140256706 A1 and any analogs thereof.

In some embodiments, the cell described herein (for example, NK cellsexpressing CARs and further including on the surface a plurality ofchemotherapeutic agent-loaded cMLVs) is administered to a subject incombination with a interleukin-15 (IL-15) polypeptide, a interleukin-15receptor alpha (IL-15Ra) polypeptide, or a combination of both a IL-15polypeptide and a IL-15Ra polypeptide e.g., hetiL-15 (AdmuneTherapeutics, LLC). hetiL-15 is a heterodimeric non-covalent complex ofIL-15 and IL-15Ra. hetiL-15 is described in, e.g., U.S. Pat. No.8,124,084, U.S. 2012/0177598, U.S. 2009/0082299, U.S. 2012/0141413, andU.S. 201110081311. In embodiments, het-IL-15 is administeredsubcutaneously. In embodiments, the subject has a cancer, e.g., solidcancer, e.g., melanoma or colon cancer. In embodiments, the subject hasa metastatic cancer.

In one embodiment, the subject can be administered an agent whichreduces or ameliorates a side effect associated with the administrationof a CAR-expressing cell. Side effects associated with theadministration of a CAR-expressing cell include, but are not limited toCRS, and hemophagocytic lymphohistiocytosis (HLH), also termedMacrophage Activation Syndrome (MAS). Symptoms of CRS include highfevers, nausea, transient hypotension, hypoxia, and the like. CRS mayinclude clinical constitutional signs and symptoms such as fever,fatigue, anorexia, myalgias, arthalgias, nausea, vomiting, and headache.CRS may include clinical skin signs and symptoms such as rash. CRS mayinclude clinical gastrointestinal signs and symptoms such as nausea,vomiting and diarrhea. CRS may include clinical respiratory signs andsymptoms such as tachypnea and hypoxemia. CRS may include clinicalcardiovascular signs and symptoms such as tachycardia, widened pulsepressure, hypotension, increased cardiac output (early) and potentiallydiminished cardiac output (late). CRS may include clinical coagulationsigns and symptoms such as elevated d-dimer, hypofibrinogenemia with orwithout bleeding. CRS may include clinical renal signs and symptoms suchas azotemia. CRS may include clinical hepatic signs and symptoms such astransaminitis and hyperbilirubinemia. CRS may include clinicalneurologic signs and symptoms such as headache, mental status changes,confusion, delirium, word finding difficulty or frank aphasia,hallucinations, tremor, dymetria, altered gait, and seizures.

Accordingly, the methods described herein can include administering thecell described herein (for example, NK cells expressing CARs andincluding on the surface a plurality of chemotherapeutic agent-loadednanoparticles such as cMLVs) to a subject and further administering oneor more agents to manage elevated levels of a soluble factor resultingfrom treatment with a CAR-expressing cell. In one embodiment, thesoluble factor elevated in the subject is one or more of IFN-γ, TNFa,IL-2 and IL-6. In an embodiment, the factor elevated in the subject isone or more of IL-1, GM-CSF, IL-10, IL-8, IL-5 and fraktalkine.Therefore, an agent administered to treat this side effect can be anagent that neutralizes one or more of these soluble factors. In oneembodiment, the agent that neutralizes one or more of these solubleforms is an antibody or antigen binding fragment thereof. Examples ofsuch agents include, but are not limited to a steroid (e.g.,corticosteroid), Src inhibitors (e.g., Dasatinib) an inhibitor of TNFa,and an inhibitor of IL-6. An example of a TNFa inhibitor is an anti-TNFaantibody molecule such as, infliximab, adalimumab, certolizumab pegol,and golimumab. Another example of a TNFa inhibitor is a fusion proteinsuch as entanercept. An example of an IL-6 inhibitor is an anti-IL-6antibody molecule or an anti-IL-6 receptor antibody molecule such astocilizumab (toe), sarilumab, elsilimomab, CNTO 328, ALD518/BMS-945429,CNTO 136, CPSI-2364, CDP6038, VX30, ARGX-109, FE301, and FM101. In oneembodiment, the anti-IL-6 receptor antibody molecule is tocilizumab. Inone embodiment, the IL-6 inhibitor is a camelid bispecific antibody thatbinds to IL6R and human serum albumin (e.g., IL6R-304-Alb8) (SEQ ID NO:2649). An example of an IL-1R based inhibitor is anakinra. In oneembodiment, an agent administered to treat the side effects ofCAR-expressing cells is a Src inhibitor (e.g., Dasatinib). In oneembodiment, an agent administered to treat the side effects ofCAR-expressing cells is the Src inhibitor Dasatinib. In embodiments,Dasatinib is administered at a dose of about 10 mg/day to 240 mg/day(e.g., 10 mg/day, 20 mg/day, 40 mg/day, 50 mg/day, 70 mg/day, 80 mg/day,100 mg/day, 110 mg/day, 120 mg/day, 140 mg/day, 180 mg/day, 210 mg/day,240 mg/day or 300 mg/day).

In one embodiment, the subject can be administered an agent whichenhances the activity of the cell described herein (for example, NKcells expressing CARs and further including on the surface a pluralityof chemotherapeutic agent-loaded nanoparticles such as cMLVs). Forexample, in one embodiment, the agent can be an agent which inhibits aninhibitory molecule. Inhibitory molecules, e.g., Programmed Death 1(PD-1), can, in some embodiments, decrease the ability of aCAR-expressing cell to mount an immune effector response. Examples ofinhibitory molecules include PD-1, PDL1, CTLA-4, TIM-3, CEACAM (e.g.,CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4 and TGFR beta. Inhibition of an inhibitory molecule, e.g., byinhibition at the DNA, RNA or protein level, can optimize aCAR-expressing cell performance. In embodiments, an inhibitory nucleicacid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA orshRNA, a clustered regularly interspaced short palindromic repeats(CRISPR), a transcription-activator like effector nuclease (TALEN), or azinc finger endonuclease (ZFN), e.g., as described herein, can be usedto inhibit expression of an inhibitory molecule in the CAR-expressingcell. In an embodiment the inhibitor is an shRNA. In an embodiment, theinhibitory molecule is inhibited within a CAR-expressing cell. In theseembodiments, a dsRNA molecule that inhibits expression of the inhibitorymolecule is linked to the nucleic acid that encodes a component, e.g.,all of the components, of the CAR. In one embodiment, the inhibitor ofan inhibitory signal can be, e.g., an antibody or antibody fragment thatbinds to an inhibitory molecule. For example, the agent can be anantibody or antibody fragment that binds to PD-1, PD-L1, PD-L2 or CTLA4(e.g., ipilimumab (also referred to as MDX-010 and MDX-101, and marketedas Yervoy®; Bristol-Myers Squibb; Tremelimumab (IgG2 monoclonal antibodyavailable from Pfizer, formerly known as ticilimumab, CP-675,206).). Inan embodiment, the agent is an antibody or antibody fragment that bindsto TIM3. In an embodiment, the agent is an antibody or antibody fragmentthat binds to CEACAM (CEACAM-1, CEACAM-3, and/or CEACAM-5). In anembodiment, the agent is an antibody or antibody fragment that binds toLAG3.

PD-1 is an inhibitory member of the CD28 family of receptors that alsoincludes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated Bcells, T cells and myeloid cells. Two ligands for PD-1, PD-L1 and PD-L2have been shown to down regulate T cell activation upon binding to PD-1.PD-L1 is abundant in human cancers. Immune suppression can be reversedby inhibiting the local interaction of PD-1 with PD-L1. Antibodies,antibody fragments, and other inhibitors of PD-1, PD-L1 and PD-L2 areavailable in the art and may be used combination with a CAR of thepresent invention described herein. For example, nivolumab (alsoreferred to as BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fullyhuman IgG4 monoclonal antibody which specifically blocks PD-1. Nivolumab(clone 5C4) and other human monoclonal antibodies that specifically bindto PD-1 are disclosed in U.S. Pat. No. 8,008,449 and WO2006/121168.Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibodythat binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonalantibodies are disclosed in WO2009/101611. Pembrolizumab (formerly knownas lambrolizumab, and also referred to as MK03475; Merck) is a humanizedIgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and otherhumanized anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,354,509and WO2009/114335. MEDI4736 (Medimmune) is a human monoclonal antibodythat binds to PDL1, and inhibits interaction of the ligand with PD1.MDPL3280A (Genentech I Roche) is a human Fe optimized IgG1 monoclonalantibody that binds to PD-L1. MDPL3280A and other human monoclonalantibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743 and U.SPublication No.: 20120039906. In other embodiments, the agent thatenhances the activity of a CAR-expressing cell is a CEACAM inhibitor(e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5 inhibitor).

In one embodiment, the agent that enhances activity of the celldescribed herein (for example, NK cells expressing CARs and furtherincluding on the surface a plurality of chemotherapeutic agent-loadednanoparticles such as cMLVs) is another agent that increases theexpression of the target antigen against which the CAR is directed. Theagents that can be administered to the subject receiving aCAR-expressing cell described herein include: Arsenic trioxide, ATRA(all-trans-retinoic acid), compounds 27, 40, 49 of, IDH2 inhibitors(e.g., AG-221) or a combination thereof. In an embodiment, the agentsare administered prior to, concurrently or after administration ofCAR-expressing cells. In preferred embodiments these agents areadministered prior to administration of CAR-expressing cells. Inpreferred embodiment, the CAR expressing cells that are administeredwith the above agents target a B cell antigen (e.g., CD19, CD20, or CD22etc.).

Cytokines that can be administered to the subject receiving the celldescribed herein (for example, NK cells expressing CARs and furtherincluding on the surface a plurality of chemotherapeutic agent-loadednanoparticles such as cMLVs) include: IL-2, IL-4, IL-7, IL-9, IL-15,IL-18, LIGHT, and IL-21, or a combination thereof. In preferredembodiments, the cytokine administered is IL-7, IL-15, or IL-21, IL12F,or a combination thereof. The cytokine can be administered once a day ormore than once a day, e.g., twice a day, three times a day, or fourtimes a day. The cytokine can be administered for more than one day,e.g. the cytokine is administered for 2 days, 3 days, 4 days, 5 days, 6days, 1 week, 2 weeks, 3 weeks, or 4 weeks. For example, the cytokine isadministered once a day for 7 days. Administration of the cytokine tothe subject that has sub-optimal response to the CAR-expressing celltherapy improves CAR-expressing cell efficacy or anti-cancer activity.In a preferred embodiment, the cytokine administered afteradministration of CAR-expressing cells is IL-7.

In one embodiment, the agent which enhances activity of the celldescribed herein (for example, NK cells expressing CARs and furtherincluding on the surface a plurality of chemotherapeutic agent-loadednanoparticles such as cMLVs) is a Brd4 inhibitor or an siRNA or an shRNAtargeting BRD4.

Pharmaceutical Composition

In various embodiments, the present invention provides a pharmaceuticalcomposition. The pharmaceutical composition includes geneticallymodified cells expressing antigen-specific CARs and having bound on thecell surface a plurality of liposomes (for example, multilamellarliposomal vesicles) or other naonparticles which encapsulate orotherwise carry one or more chemotherapeutic agents; and anypharmaceutically acceptable excipient. In exemplary embodiments, thegenetically modified cells are NK cells that express CARs specific toHer2 and/or CD19 and are surface bonded with a plurality ofnanoparticles (e.g., multilamellar liposomal vesicles) which encapsulatepaclitaxel.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients may be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous. Examples of excipients include but are notlimited to starches, sugars, microcrystalline cellulose, diluents,granulating agents, lubricants, binders, disintegrating agents, wettingagents, emulsifiers, coloring agents, release agents, coating agents,sweetening agents, flavoring agents, perfuming agents, preservatives,antioxidants, plasticizers, gelling agents, thickeners, hardeners,setting agents, suspending agents, surfactants, humectants, carriers,stabilizers, and combinations thereof.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, oral, transmucosal, transdermal, parenteral or enteral.“Parenteral” refers to a route of administration that is generallyassociated with injection, including intraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions may be in the form of solutions or suspensions for infusionor for injection, or as lyophilized powders. Via the parenteral route,the compositions may be in the form of solutions or suspensions forinfusion or for injection. Via the enteral route, the pharmaceuticalcompositions can be in the form of tablets, gel capsules, sugar-coatedtablets, syrups, suspensions, solutions, powders, granules, emulsions,microspheres or nanospheres or lipid vesicles or polymer vesiclesallowing controlled release. Typically, the compositions areadministered by injection. Methods for these administrations are knownto one skilled in the art.

The pharmaceutical compositions according to the invention can containany pharmaceutically acceptable carrier. “Pharmaceutically acceptablecarrier” as used herein refers to a pharmaceutically acceptablematerial, composition, or vehicle that is involved in carrying ortransporting a compound of interest from one tissue, organ, or portionof the body to another tissue, organ, or portion of the body. Forexample, the carrier may be a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of theformulation. It must also be suitable for use in contact with anytissues or organs with which it may come in contact, meaning that itmust not carry a risk of toxicity, irritation, allergic response,immunogenicity, or any other complication that excessively outweighs itstherapeutic benefits.

The pharmaceutical compositions according to the invention can also beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols and water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulation, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or non-aqueous suspension. Such a liquid formulation may beadministered directly p.o. or filled into a soft gelatin capsule.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

Before administration to patients, formulants may be added to theengineered immune effector cell, or a population of cells containing aplurality of the engineered immune effector cell. A liquid formulationmay be preferred. For example, these formulants may include oils,polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin,surfactants, bulking agents or combinations thereof.

Carbohydrate formulants include sugar or sugar alcohols such asmonosaccharides, disaccharides, or polysaccharides, or water solubleglucans. The saccharides or glucans can include fructose, dextrose,lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran,pullulan, dextrin, alpha and beta cyclodextrin, soluble starch,hydroxethyl starch and carboxymethylcellulose, or mixtures thereof.“Sugar alcohol” is defined as a C4 to C8 hydrocarbon having an —OH groupand includes galactitol, inositol, mannitol, xylitol, sorbitol,glycerol, and arabitol. These sugars or sugar alcohols mentioned abovemay be used individually or in combination. There is no fixed limit toamount used as long as the sugar or sugar alcohol is soluble in theaqueous preparation. In one embodiment, the sugar or sugar alcoholconcentration is between 1.0 w/v % and 7.0 w/v %, more preferablebetween 2.0 and 6.0 w/v %.

Amino acids formulants include levorotary (L) forms of carnitine,arginine, and betaine; however, other amino acids may be added.

In some embodiments, polymers as formulants include polyvinylpyrrolidone(PVP) with an average molecular weight between 2,000 and 3,000, orpolyethylene glycol (PEG) with an average molecular weight between 3,000and 5,000.

It is also preferred to use a buffer in the composition to minimize pHchanges in the solution before lyophilization or after reconstitution.Most any physiological buffer may be used including but not limited tocitrate, phosphate, succinate, and glutamate buffers or mixturesthereof. In some embodiments, the concentration is from 0.01 to 0.3molar. Surfactants that can be added to the formulation are shown in EPNos. 270,799 and 268,110.

Another drug delivery system for increasing circulatory half-life is theliposome. Methods of preparing liposome delivery systems are discussedin Gabizon et al., Cancer Research (1982) 42:4734; Cafiso, BiochemBiophys Acta (1981) 649:129; and Szoka, Ann Rev Biophys Eng (1980)9:467. Other drug delivery systems are known in the art and aredescribed in, e.g., Poznansky et al., DRUG DELIVERY SYSTEMS (R. L.Juliano, ed., Oxford, N.Y. 1980), pp. 253-315; M. L. Poznansky, PharmRevs (1984) 36:277.

Kits

In various embodiments, the present invention provides kits comprisingthe pharmaceutical compositions described herein.

The kit is an assemblage of materials or components, including at leastone of the inventive vectors and compositions. Thus, in some embodimentsthe kit contains a composition that has genetically modified cellsexpressing antigen-specific CARs and having bound on the cell surface aplurality of liposomes (for example, multilamellar liposomal vesicles)or other nanoparticles which encapsulate or otherwise carry one or morechemotherapeutic agents, as described above.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. In one embodiment, the kit isconfigured particularly for human subjects. In further embodiments, thekit is configured for veterinary applications, treating subjects suchas, but not limited to, farm animals, domestic animals, and laboratoryanimals.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to treat, reduce the severity of, inhibit or prevent cancer in asubject. Optionally, the kit also contains other useful components, suchas, measuring tools, diluents, buffers, pharmaceutically acceptablecarriers, syringes or other useful paraphernalia as will be readilyrecognized by those of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example, the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well-knownmethods, preferably to provide a sterile, contaminant-free environment.As used herein, the term “package” refers to a suitable solid matrix ormaterial such as glass, plastic, paper, foil, and the like, capable ofholding the individual kit components. Thus, for example, a package canbe a glass vial used to contain suitable quantities of a composition.The packaging material generally has an external label which indicatesthe contents and/or purpose of the kit and/or its components.

EXAMPLES

The following examples are not intended to limit the scope of the claimsto the invention, but are rather intended to be exemplary of certainembodiments. Any variations in the exemplified methods which occur tothe skilled artisan are intended to fall within the scope of the presentinvention.

Example 1

Experimental Methods

Cell Lines and Reagents:

MDA.MB.468 (ATCC HTB-132) and SKOV3 (ATCC HTB-77) tumor cell lines weremaintained in a 5% CO2 environment in RPMI 1640 (Gibco) mediasupplemented with 10% FBS, 1% pen-strep, and 2 mM L-glutamine. NK92cells (Dr. Jihane Khalife, Children's Hospital Los Angeles, ATCCCRL-2407) were maintained in MEM-α (Gibco) supplemented with 10% FBS,10% horse serum, 1% NEAA, 1% pen-strep, 1% sodium pyruvate, 0.1 mM 2-βmercaptoethanol, 0.2 mM myo-inositol, and 2.5 μM folic acid. CD19⁺ SKOV3(SKOV.CD19) cells were generated by transducing SKOV3 cells withlentivirus containing CD19 cDNA and sorting CD19⁺ cells withfluorescence-activated cell sorting (FACS).

PTX was purchased from Sigma-Aldrich (St. Louis, Mo.). All lipids werepurchased from NOF Corporation (Japan):1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dioleoyl-sn-glycero-3-phospho-(10-rac-glycerol) (DOPG), and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)but-yramide(maleimide-headgroup lipid, MPB-PE).

Synthesis of Nanoparticles:

Liposomes were prepared based on the conventionaldehydration-rehydration method (Joo, K, et al. (2013). Crosslinkedmultilamellar liposomes for controlled delivery of anticancer drugs.Biomaterials 34: 3098-3109; Moon, J, et al. (2011).Interbilayer-crosslinked multilamellar vesicles as synthetic vaccinesfor potent humoral and cellular immune responses. Nat Mater 10:243-251). cMLVs were prepared from 1.5 μmol of lipidsDOPC:DOPG:MPE-PE=40:10:50 mixed in chloroform and evaporated under argongas before drying under a vacuum overnight to form dried lipid films.The lipid film was rehydrated in 10 mM Bis-Tris propane at pH 7.0. Afterthe lipid was mixed through vigorous vortexing every 10 minutes for 1hour, they underwent three cycles of 15-second sonication (MisonixMicroson XL2000, Farmingdale, N.Y.) and rested on ice at 1-minuteintervals after each cycle. A final concentration of 10 mM MgCl₂ wasadded to induce divalent-triggered vesicle fusion. The crosslinking ofmultilamellar vesicles (cMLVs) was performed by addition ofDithiothreitol (DTT, Sigma-Aldrich) at a final concentration of 1.5 mMfor 1 h at 37° C. The cMLVs were collected by centrifugation at 14,000 gfor 5 minutes and washed twice with PBS. The particles were suspended infiltered water, vortexed and sonicated prior to analysis. Morphologicalanalysis of the multilamellar structure of vesicles was performed andconfirmed by cryo-electron microscopy as previously studied by Joo, K.,et al. The hydrodynamic size of cMLVs was measured by dynamic lightscattering (Wyatt Technology, Santa Barbara, Calif.).

In Vitro Drug Encapsulation and Release:

The amount of incorporated paclitaxel in the cMLV(PTX) was determined byC-18 reverse-phase high-performance liquid chromatography (RPHPLC)(Beckman Coulter, Brea, Calif.). The cMLV(PTX) suspension was diluted byadding water and acetonitrile to a total volume of 0.5 mL. Extraction ofpaclitaxel was accomplished by adding 5 mL of tert-butyl methyl etherand vortex-mixing the sample for 1 min. The mixtures were centrifuged,and the organic layer was transferred into a glass tube and evaporatedunder argon. Buffer A (95% water, 5% acetonitrile) was used to rehydratethe glass tube. To test PTX concentration, 1 mL of the solution wasinjected into a C18 column, and the paclitaxel was detected at 227 nm(flow rate 1 mL/min). To obtain the release kinetics of PTX from cMLVsbefore and after cell conjugation, cMLV(PTX) and CAR.NK.cMLV(PTX) wereincubated in 10% FBS-containing media at 37° C. and were spun down andresuspended with fresh media daily. The PTX was quantified from theremoved media by HPLC every day.

Nanoparticle Conjugation with Cells and In Situ PEGylation:

Chemical conjugation of cMLVs to the cells was performed based on amethod provided in previous studies (Huang, B, et al. (2015). Activetargeting of chemotherapy to disseminated tumors usingnanoparticle-carrying T cells. Sci Transl Med 77: 291ra294; Stephan, M,et al (2010). Therapeutic cell engineering with surface-conjugatedsynthetic nanoparticles. Nature Med 16: 1035-1041). We resuspended10×10⁶ cells/mL in serum free MEM-α (Gibco) medium. Equal volumes ofnanoparticles were resuspended in nuclease free water at different cMLVto NK cell conjugation ratios and incubated at 37° C. The cells andnanoparticles were mixed every 10 minutes for 30 minutes. After a PBSwash to remove unbound cMLVs from cells, cells were further incubatedwith 1 mg/ml thiol-terminated 2-kDa PEG at 37° C. for 30 minutes inmedia to quench residual maleimide groups on cell-bound particles. Weperformed two PBS washes to remove unbound PEG. For quantification ofcell bound particles, particles were fluorescently labeled with thelipid-like fluorescent dye DiD (Invitrogen). Particle fluorescence wasdetected with flow cytometry and a fluorescent microplate reader. cMLVswere labeled with the lipid-like dye DiD and CAR.NK cells were stainedwith carboxyfluorescein diacetate succinimide ester (CF SE)(Invitrogen), which allowed the conjugation of cMLVs to NK cells to beeasily detected using confocal microscopy.

Lentiviral and Retroviral Production and Transduction of NK92 Cells:

Our anti-Her2 CAR construct (Zhao, Y, et al. (2009). A Herceptin-basedchimeric antigen receptor with modified signaling domains leads toenhanced survival of transduced T lymphocytes and antitumor activity. JImmunol 183: 5563-5574) was cloned into a lentiviral pCCW vector (a pCCLvector (Haas, D, et al (2003). The moloney murine leukemia virusrepressor binding site represses expression in murine and humanhematopoietic stem cells. J Virol 77: 9439-9450; Dull, T, et al (1998).A third-generation lentivirus vector with a conditional packagingsystem. J Virol 72: 8463-8471; Logan, A, et al (2004) Factorsinfluencing the titer and infectivity of lentiviral vectors. Hum GeneTher 15: 976-988)) with an additional WRE posttranscriptional regulatoryregion. The CAR consisted of the anti-Her2 scFv 4D5, a CD8 hinge andtransmembrane region, and CD28, 4-1BB, and CD3ζ cytoplasmic regions. Ouranti-CD19 CAR construct was cloned into a MP-71 retroviral vectorbackbone (Engels, B, et al. (2003) Retroviral vectors for high-leveltransgene expression in T lymphocytes. Hum Gene Ther 14: 1155-1168) andcontained an anti-CD19 scFv, a CD8 hinge and transmembrane region, andCD28 and CD3ζ cytoplasmic regions. These plasmids were used to transfectHEK 293T cells in 30 mL plates using CaCl2 precipitation methods. Freshmedia (high glucose DMEM supplemented with 10% FBS and 1% pen-strep) wasplated onto the cells 4 hours after initial transfection. Supernatantswere harvested and filtered (0.45 μm) 48 hours later. NK92 cells weretransduced with fresh retrovirus. Lentiviral supernatant wasconcentrated (25,000 rpm for 90 minutes at 4° C.), resuspended in HBSS,and frozen at −80° C. until later use. NK92 cells were transduced withconcentrated lentivirus at MOI 40; the titer was based on transductionof 293T cells.

CAR Detection on T Cell Surface:

Three days after transduction, anti-CD19 CAR.NK cells (1×10⁵) wereincubated with biotinylated Protein L (Peprotech) at a volume ratio of1:50 in PBS+4% FBS at 4° C. for 45 minutes and rinsed with PBS. Thecells were subsequently incubated with streptavidin conjugated to FITC(Biolegend) at a volume ratio of 1:500 in PBS+4% FBS at 4° C. for 10minutes, rinsed twice, and read using flow cytometry. Anti-Her2 CAR.NKcells (1×10⁵) were incubated with rhHer2-Fc chimera (Peprotech) at avolume ratio of 1:50 (2 μg/mL) in PBS at 4° C. for 30 minutes and rinsedwith PBS. The cells were subsequently incubated with PE-labeled goatanti-human Fc (Jackson ImmunoResearch) at a volume ratio of 1:150 in PBSat 4° C. for 10 minutes, rinsed, and read using flow cytometry.Nontransduced NK cells served as a negative control.

Internalization Assay:

Quantification of cell cMLVs internalization was performed based on amethod previously described (Huang, B, et al (2015). Active targeting ofchemotherapy to disseminated tumors using nanoparticle-carrying T cells.Sci Transl Med 77: 291ra294; Stephan, M, et al (2010). Therapeutic cellengineering with surface-conjugated synthetic nanoparticles. Nature Med16: 1035-1041). NK and CAR.NK cells were conjugated with 5 mole % 18:1PE CF(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein)(ammonium salt) (Avanti, Polar Lipids)-tagged liposomes. After 2 PBSwashes, cells were transferred to fibronectin (10ug/ml)-coated 96 wellplates. After a 2 hour incubation time, half of the wells were treatedwith 100 μl trypan blue in HBSS (0.25 mg/mL), an extracellularfluorescence quenching dye, for 1 min in order to differentiate betweenmembrane-bound and internalized liposomes. Trypan blue was removed bygentle vacuum aspiration and the cell uptake of liposomes was quantifiedby a fluorescence plate reader.

Cytokine Release Assay:

NK cells (1×10⁵ per well) were coincubated with target cells in 96-wellplates at a 1:1 ratio for 6 hours at 37° C. 1 μg Brefeldin-A (Sigma) wasadded to each well to prevent protein transport. At the end of theincubation, cells were permeabilized using the CytoFix/CytoPerm kit (BDBiosciences) and stained for CD8 and IFN-γ using Pacific Blue-conjugatedanti-human CD8 (Biolegend) and PE-conjugated anti-human IFN-γ(Biolegend). Unstimulated cells served as a negative control. Resultswere read using flow cytometry.

Cytotoxicity Assay:

Target cells (1×10⁴) were labeled with 5 μM carboxyfluoresceinsuccinimidyl ester (CF SE, Life Technologies) as previously described(Han, X, et al. (2017). Masked chimeric antigen receptor fortumor-specific activation. Molecular Therapy 25: 274-284) andcoincubated with NK cells at various ratios in 96-well plates for 24hours at 37° C. The cells were then incubated in 7-AAD (LifeTechnologies) in PBS (1:1000 dilution) for 10 minutes at roomtemperature and analyzed via flow cytometry. Percentages of killed cellswere calculated as [CFSE⁺7-AAD⁺ cells/(CFSE⁺7-AAD⁻+CFSE⁺7-AAD⁺)] cells,with live/dead gates based on control wells of target cells to accountfor spontaneous cell death.

NK92 and SKOV3 cells were seeded in 96-well plates at 2×10⁴ cells perwell in 10% FBS-containing media and grown at 37° C. in the presence of5% CO2 for 6 hours. Cells were incubated with various concentrations ofcMLV (PTX) as previously described (Liu, Y, et al (2014). Codelivery ofdoxorubicin and paclitaxel by cross-linked multilamellar liposomeenables synergistic antitumor activity. Mol Pharm 11: 1651-1661) andcell viability was assessed using the Cell Proliferation Kit II (XTTassay) from Roche Applied Science (Indianapolis, Ind.) according to themanufacturer's instructions. Cell viability percentage was determined bysubtracting absorbance values obtained from media-only wells from thetreated wells and then normalized by the control wells containing cellswithout drugs.

Transmigration Assay:

NK cell transmigration assays were performed in 24 mm diameter 3 μm poresize Transwell plates (Costar). NK cells either conjugated orunconjugated to cMLVs were plated on the upper wells and media was addedto the lower wells. The chemoattractant CXCL9 (0.1 mg/ml, Peprotech) wasadded to the lower wells. After incubation at 37° C. for 6 hours, NKcells that had migrated into the lower chamber were counted.

In Vivo Biodistribution Study:

Female 6-10 weeks-old NOD.Cg-Prkdc^(scid)IL2Rγ^(tmlWjl)/SZ (NSG) micewere purchased from Jackson Laboratories (Bar Harbor, Me.). All micewere held under specific pathogen-reduced conditions in the animalfacility of the University of Southern California (Los Angeles, Calif.,USA). All experiments were performed in accordance with the guidelinesset by the National Institute of Health and the University of SouthernCalifornia on the Care and Use of Animals. A total of 3.5×10⁶ SKOV3.CD19cells were inoculated subcutaneously into the flanks ofNOD/scid/IL2rγ−/− (NSG) mice on Day −14, and tumors were allowed to growuntil they reached 100 mm³. On Day 0, mice were injected intravenouslythrough the tail vein with either cMLV(DiD) or CAR.NK.cMLV(DiD). 24, 48,and 72 hours after injection, mice were sacrificed and organs wereanalyzed for fluorescence intensity. DiD tissue fluorescence for eachorgan was quantified using the IVIS Spectrum imaging system and thepercentage of injected dose per gram of tissue (% ID/g) was calculated.

Xenograft Tumor Model:

A total of 3.5×10⁶ SKOV3.CD19 cells were inoculated subcutaneously intothe flanks of NSG mice on Day −14, and tumors were allowed to grow to70-100 mm³. Mice were randomly divided into six groups of five miceeach. On Days 0, 4, 7, and 11, the mice were injected intravenouslythrough the tail vein with either PBS, cMLV(PTX) only, nontransduced NKcells only, CAR.NK cells only, mixed CAR.NK+cMLV(PTX) which were notconjugated together, or conjugated CAR.NK.cMLV(PTX). 5×10⁶ cells permouse were injected each time in the groups that were given NK cells.Tumor growth and body weight of the mice were recorded until sacrifice.The tumor length and width were measured with a fine caliper, and tumorvolume was calculated as ½×(length)×(width)².

Intratumoral PTX Concentration Measurements Ex Vivo:

Using high performance liquid chromatography (HPLC), the PTXconcentration in the frozen tumor tissues was quantified as previouslydetailed (Liu, Y, et al (2014). Codelivery of chemotherapeutics viacrosslinked multilamellar liposomal vesicles to overcome multidrugresistance in tumor. PLoS ONE 9: e110611). Briefly, thawed tumor tissueswere chopped and homogenized in ethyl acetate, with a knownconcentration of docetaxel added to each sample as an internal standard.The samples were centrifuged and the organic layer was transferred to aclean tube. The organic layer was evaporated under a stream of argon andrehydrated in diluted acetonitrile. After running the samples on HPLC,the peak heights were analyzed to determine intratumoral PTXconcentration.

Immunohistochemistry of Tumors, Cardiac Toxicity, and Confocal Imaging:

Tumors were excised, fixed, frozen, cryo-sectioned, and mounted ontoglass slides. Frozen sections were fixed and rinsed with cold PBS. Afterblocking and permeabilization, the slides were washed with PBS andincubated with a terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL) reaction mixture (Roche, Indianapolis, Ind.) for 1 hourand counterstained with 4′,6-diamidino-2-phenylindole (DAPI)(Invitrogen, Carlsbad, Calif.). Fluorescence images were acquired by aYokogawa spinning-disk confocal scanner system (Solamere TechnologyGroup, Salt Lake City, Utah) using a Nikon Eclipse Ti-E microscope.Illumination powers at 405, 491, 561, and 640 nm solid-state laser lineswere provided by an AOTF (acousto-optical tunable filter)-controlledlaser-merge system with 50 mW for each laser. All images were analyzedusing Nikon NIS-Elements software. For quantifying TUNEL positive cells,four regions of interest (ROI) were randomly chosen per image at 10×magnification. Within one region, the area of TUNEL-positive nuclei andthe area of nuclear staining were counted by Nikon NIS-Element software,with data expressed as % total nuclear area stained by TUNEL in theregion. For cardiac toxicity, heart tissues were harvested 2 days afterthe last injection and were fixed in 4% formaldehyde. The tissues werefrozen and then cut into sections and mounted onto glass slides. Thefrozen sections were stained with hematoxylin and eosin. Histopathologicspecimens were examined by EVOS light microscopy.

Statistics:

The differences between two groups were determined with Student's ttest. The differences among three or more groups were determined with aone-way analysis of variance (ANOVA).

Example 2 Anti-CD19 and Anti-Her2 CARs are Expressed in NK92 Cells

We confirmed the ability of NK92 cells to express anti-CD19 andanti-Her2 CARs, which consisted of an scFv-derived antigen bindingdomain, CD8 hinge and transmembrane region, CD28 and/or 4-1BBcostimulatory domains, and CD3ζ signaling domain. Anti-CD19 CAR.NK cellswere generated with retroviral transduction using the previouslydocumented MP71 vector generously provided by Dr. Wolfgang Uckert. Theanti-Her2 CAR.NK cells were generated with lentiviral transduction usinga previously described trastuzumab-derived CAR in a pCCW vector, whichis based off the pCCL vector⁴³⁻⁴⁵ with an added WRE posttranscriptionalregulatory region. Transduced cells were sorted using fluorescenceactivated cell sorting to further increase the percentage of CAR⁺ cells(FIG. 1E). CAR expression was stable several months after initialtransduction and sorting.

cMLVs are Stably Conjugated to the NK Cell Surface

Previous studies have shown that cross-linked multilamellar liposomalvesicles (cMLVs) were successfully incorporated with both hydrophobicand hydrophilic drugs. These vesicles were synthesized throughcovalently crosslinking functionalized headgroups of adjacent lipidbilayers using the conventional dehydration-rehydration method.Synthesized cMLV nanoparticles were stably conjugated to the reducedthiol groups present on the surface of NK cell via the thiol-reactivemaleimide headgroups present on the lipid bilayer surface. High levelsof free thiols were detected on the surfaces of lymphocytes. Theconjugation was performed in two steps. First, NK cells and cMLVs werecoincubated to induce particle coupling to free thiols on the cellsurface. After the initial reaction, the cMLV-conjugated cells underwentin situ PEGylation to quench residual thiol reactive groups. Todetermine the maximum numbers of particles that could be conjugated perNK cell, we performed a serial dilution of the conjugation at differentfluorescent-labeled cMLVs to cell ratios (2000:1, 1000:1, 500:1, 100:1,and 10:1). Between the conjugation ratio of 2000:1 and 1000:1, thenumber of conjugated liposomes per cell began to plateau and showed anaverage conjugation of approximately 150 nanoparticles per cell (FIG.1B). From this data, we determined that the optimal ratio to use was1000:1, as further increasing it did not increase the number ofconjugated cMLVs on the cell surface. We used this ratio for allsubsequent experiments. Confocal imaging was used to confirm andvisualize the conjugation of cMLVs to the NK cell surface (FIG. 1C, FIG.1F).

The major advantages of extended surface retention of nanoparticles onthe surface of carrier cells are as follows: (1) prevention fromimmediate particle degradation due to internalization into degradativeintracellular compartments and (2) sustained drug release from theparticle-conjugated cells which allows for effective targeting of thedrug to tumor cells. Nanoparticles can be endocytosed by a variety ofcells, including endothelial cells and macrophages. However, for ourstudy, it is crucial that the cMLVs remain on the NK cell surface. Toaddress this, we performed an experiment to determine theinternalization of these particles after conjugation. To determinewhether these NK cells could also trigger liposome endocytosis, weconjugated NK cells with cMLVs tagged with a PE CF fluorescein dye, thenwarmed the cells to 37° C. and assessed cell-associated fluorescenceover time. Attachment of cMLVs to NK cells did not trigger cell uptakeof these particles and particles bound to NK cells remained at the cellsurface as shown in FIG. 1D.

CAR.NK Cells have Greater Cytotoxic Effects Against Antigen-ExpressingTarget Cells In Vitro and are Less Sensitive to PTX

We assessed the ability of CAR.NK cells to trigger cytotoxic effectsagainst the appropriate antigen-expressing target cells by coincubatingnontransduced NK or CAR.NK cells with various target cell lines andreading the results with flow cytometry. We used lentivirus to transduceSKOV3 cells to express CD19 (SKOV.CD19) to serve as target cells for ouranti-CD19 CAR.NK cells. Both CD19 and Her2-targeting CAR.NK cellsdemonstrated significantly greater cytotoxicity against theantigen-expressing target cells (SKOV.CD19 and SKOV3, respectively)compared with either nontransduced NK cells or CAR.NK cells coincubatedwith target cells that did not express the cognate antigen (SKOV3 andMDA.MB.468, respectively, FIG. 2A, FIG. 2B). These trends were observedat all effector-to-target ratios (p<0.01 for 1:1 and 10:1, p<0.001 for5:1) and indicated CAR-mediated cell killing.

Since NK92 cells originate from a patient with NK cell lymphoma, theseallogenic cells are irradiated prior to clinical use to prevent themfrom proliferating in vivo. Irradiation did not affect the cytotoxiccapabilities of our CAR.NK cells (FIG. 2C). We also performed a cellviability assay to demonstrate that SKOV3 cells were more sensitive toPTX than NK cells were (FIG. 2D). This ensures that the NK cells cancarry enough PTX to kill target cells without succumbing to PTX-inducedtoxicity themselves. cMLVs conjugated to NK cells also release themajority of their PTX payload by Day 3 (FIG. 2E).

CAR.NK Function is Unaffected by cMLV Conjugation and Enhanced withcMLV(PTX) Conjugation In Vitro

We ensured that the conjugation of cMLVs to the CAR.NK cell surface doesnot affect the functionality of the CAR.NK cell itself. To detect NKcell activation upon antigen binding, we performed an IFN-γ releaseassay, coculturing various target cell lines with NK cells with orwithout cMLV conjugation. None of the CAR.NK cells reacted whenincubated alone or when cocultured with target cells without the cognateantigen, but coincubation with the correct antigen-expressing targetcells resulted in significantly greater percentages of IFN-γ⁺ cells fromboth anti-CD19 and anti-Her2 CAR.NK cell lines (p<0.05) demonstratingspecificity towards the appropriate TAA. When the CAR.NK cells wereconjugated to either empty cMLVs containing no drug (CAR.NK.cMLV(EMPTY))or PTX-loaded cMLVs (CAR.NK.cMLV(PTX)), IFN-γ release was notsignificantly different from that of unconjugated CAR.NK cells (FIG. 3A,FIG. 3B).

We repeated the cytotoxicity assays using an effector-to-target ratio of1:1 with CAR.NK cells that were unconjugated, conjugated to empty cMLVs(CAR.NK.cMLV(EMPTY)), or conjugated to PTX-loaded cMLVs(CAR.NK.cMLV(PTX)). CAR.NK.cMLV(EMPTY) did not have significantlyaffected cell killing, but cytotoxicity against target cells wassignificantly increased with CAR.NK.cMLV(PTX) (FIG. 3C, FIG. 3D). Thesedata indicate that while empty cMLVs do not affect CAR.NK function, therelease of PTX from cMLVs in proximity to the target cells furtherboosted cytotoxic effects.

Finally, we monitored NK migration with or without cMLV conjugation. Inorder to affect an antitumor response, NK cells must extravasate intoand migrate within the tumor site in response to chemoattractants. Toensure that cMLV conjugation to the NK surface did not impact cellmigration, we performed NK cell transmigration assays. Thechemoattractant CXCL9 was used to promote NK cell migration to the lowerchamber of the wells. There were significantly more migrated NK cells inthe lower chamber when CXCL9 was used as an attractant compared to theplain media control (p<0.05), but there was no significant differencebetween conjugated and unconjugated groups, indicating that conjugationof cMLVs to the cell surface did not impact NK migratory abilities (FIG.3E).

CAR.NK.cMLV Enhances Delivery of cMLVs to the Tumor Site

After confirming the functionality of our cMLV(PTX)-conjugated CAR.NKcells in vitro, we performed a biodistribution study to determine ifCAR.NK cells enhanced cMLV homing to the tumor site. The fluorescent dyeDiD was used to tag cMLVs (cMLV(DiD)) and track their presence invarious organs. NSG mice were subcutaneously injected with SKOV.CD19cells. Two weeks after tumor inoculation, mice were intravenouslyinjected with cMLV(DiD) or conjugated CAR.NK.cMLV(DiD). Mice weresacrificed and organs were analyzed for fluorescence signal at varioustime points. At 24 hours (FIG. 4A, FIG. 4B), most of the cMLVs from bothgroups were still circulating in the blood. The CAR.NK.cMLV(DiD) grouphad significantly more cMLVs in the blood (p<0.001), lymph node(p<0.05), and tumor (p<0.01), while the cMLV(DiD) group hadsignificantly more accumulation in the liver (p<0.001). At 48 hours(FIG. 4C, FIG. 4D), most of the cMLV(DiD) group had accumulated in theliver, but the CAR.NK.cMLV(DiD) group had significantly more cMLVs inthe blood, lymph node, spleen, and tumor (p<0.001). By 72 hours (FIG.4E, FIG. 4F), most of the cMLV(DiD) signal was gone, with only smallamounts detectable in the liver, blood, and tumor. In contrast, theCAR.NK.cMLV(DiD) group had significantly more cMLVs in the blood, lymphnode, spleen, and tumor (p<0.001). Overall, cMLV(DiD) without a cellacting as a chaperone were likely cleared by the liver, as hepaticclearance serves as the main clearance route for particles too large tobe cleared by the kidneys. In contrast, cMLV(DiD) that were conjugatedto CAR.NK cells were able to home to the tumor site.

CAR.NK.cMLV(PTX) Enhances Antitumor Efficacy In Vivo

We established a mouse xenograft model to observe the effects of theanti-CD19 CAR.NK cells in vivo. NSG mice were subcutaneously injectedwith SKOV.CD19 cells. Two weeks after tumor inoculation, mice wererandomly divided into six groups and injected via tail veins(intravenously) with (1) PBS as a control, (2) cMLV(PTX) only, withoutany cellular component, (3) nontransduced NK cells only, (4) CAR.NKcells only, (5) mixed cMLV(PTX)+CAR.NK which were coinjected but notconjugated, and (6) conjugated CAR.NK.cMLV(PTX) cells. In group (6), 0.1mg PTX was injected per mouse. For a 20 g mouse, 5 million cells wereadministered per injection, for a total of four injections. Mice treatedwith CAR.NK.cMLV(PTX) had significantly slowed tumor growth compared toPBS, cMLV(PTX), and NK groups (p<0.001), and significantly slowed tumorgrowth compared to CAR.NK and CAR.NK+cMLV(PTX) groups as well (p<0.01,FIG. 5). These data support the hypothesis that both immunotherapeuticeffects from the NK cells and chemotherapeutic effects from the PTX playa role in the killing of tumor cells, as either component alone was notas effective as when the two were combined. Furthermore, even the micetreated with CAR.NK+cMLV(PTX) did not have as great an antitumorresponse as did the mice treated with CAR.NK.cMLV(PTX). Thisdemonstrates that the conjugation between the drug and the NK cell iscrucial to receiving the full benefits of the treatment system, and thatthe CAR.NK cells are facilitating the delivery of PTX to the tumor sitefor enhanced anticancer effects.

CAR.NK.cMLV(PTX) Enhances PTX Delivery into Tumor Site

We performed ex vivo analysis of our mouse xenograft tumor model tosupport our hypothesis that CAR.NK cells facilitate PTX delivery intothe tumor site. Using high performance liquid chromatography (HPLC), wequantified the intratumoral PTX concentrations in mice treated with PTX,including the groups cMLV(PTX), CAR.NK+cMLV(PTX), and CAR.NK.cMLV(PTX).The conjugated group, CAR.NK.cMLV(PTX), had significantly higher PTXconcentrations within the tumor tissue compared to the cMLV(PTX) orCAR.NK+cMLV(PTX) (p<0.01 and p<0.001, respectively, FIG. 6A).

We also used confocal imaging to visualize apoptotic cells in tumortissues fixed on glass slides. There were more terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL)⁺ cells in groups treated withCAR.NK cells than in control or NK cell groups, indicating greater cellkilling from CAR.NK cells, as shown in FIG. 6B. A higher level of cellapoptosis was observed in the group treated with CAR.NK+cMLV(PTX) whencompared to cMLV(PTX) treatment only, but the degree of cell apoptosiswas similar when compared to CAR.NK treatment. Notably,CAR.NK.cMLV(PTX)-treated tumors had the greatest degree of cellapoptosis, indicating synergistic efficacy induced by the co-localizeddelivery of the drug and CAR.NK cells.

Finally, as the therapeutic effect of PTX is limited by itscardiotoxicity, slices of fixed heart tissue stained with hematoxylinand eosin were imaged with light microscopy. Cardiotoxicity was definedas myofibrillary loss and disarray, as well as cytoplasmicvacuolization. We observed no damage to the cardiac tissues in any ofthe treatment groups (FIG. 6C). Since our delivery was targeted, we wereable to use a very low dose of PTX (0.5 mg/kg) compared to those used inconventional PTX-based treatments⁵⁷⁻⁵⁹, thus resulting in minimalcardiotoxicity.

Our system combines nanoparticle-based drug delivery with immunotherapyto produce a cell-mediated, active targeting strategy. In vitro, wedemonstrate that cMLV conjugation to NK cells does not triggerendocytosis, even though NK cells have phagocytotic capabilities. Theparticles remain on the NK cell surface, perhaps in part due to the sizeof the cMLVs—previous studies have shown that surface-conjugatedparticles larger than 50 nm in diameter are not efficientlyinternalized. Furthermore, the covalent linkage ofmaleimide-functionalized cMLVs to free thiols on immune cell surfaceshas been shown to be stable for days after initial conjugation and evenafter cell division. While the exact mechanisms of this prolongedsurface retention remain to be discovered, the maleimide-thiolconjugation strategy has been shown to be a promising method of immunecell surface engineering.

We also have demonstrated in vitro that CAR.NK cells can specificallykill antigen-expressing cancer cells, that cMLV conjugation does notadversely affect NK cell function, and that conjugation of cMLV(PTX) toCAR.NK cells further augments cytotoxicity. While many studies of CAR.NKcells include results from cytotoxicity assays but not from cytokinerelease assays, we show that CAR.NK cells release IFN-γ in response toTAA⁺ target cells. Neither CAR.NK cells coincubated with TAA⁻ targetcells nor nontransduced NK cells coincubated with any target cellsrelease IFN-γ. These results indicate that the enhanced cytotoxicity ofCAR.NK cells was accompanied by an increase in IFN-γ release. Inaddition to sensitizing tumor cells to NK cytotoxicity, IFN-γ release byboth primary NK cells and NK cell lines signals to surrounding immunecells, including T cells, dendritic cells, monocytes, and macrophages,initiating broader adaptive and innate immune responses.

Our in vivo biodistribution study further supports that CAR.NK cellsenhance nanoparticle accumulation within the tumor site. Mice treatedwith cMLV(DiD) without a cell chaperone had significantly greater cMLVaccumulation in the liver, likely indicating hepatic clearance ascommonly observed with larger liposomes. However, theCAR.NK.cMLV(DiD)-treated mice had significantly greater cMLVaccumulation at the tumor site. Additionally, significantly highersignal was observed in organs to which NK cells naturally home, such asthe spleen and lymph nodes. Our in vivo and ex vivo data provideevidence that CAR.NK cells facilitate the delivery of thechemotherapeutic drug PTX to the tumor site, slowing tumor growth andincreasing intratumoral PTX concentrations more effectively than anyother treatment group, including coadministered but not conjugatedCAR.NK and cMLV(PTX). Finally, we were able to use a low dose of PTX anddid not observe any cardiotoxicity.

We found that certain doses of PTX can kill tumor cells but not NK92cells, creating a therapeutic window in which we can use NK92 cells todeliver this chemotherapeutic drug to kill tumor cells but not thecarrier cells. However, we do not believe that this system is limited toPTX delivery. For example, murine T cells have been shown to deliver theanticancer drug SN-38 to lymphoma sites in vivo using drug-loadednanocapsules conjugated to the cell surface. SN-38 effectively killedlymphoma cells but was not toxic to the T cell carriers. Another studydemonstrated that primary human T cells can enhance antitumor immuneresponses using surface-conjugated liposomes carrying theproinflammatory cytokines IL-15 and IL-21. Surface engineering of immunecells has allowed a number of drugs or adjuvants to be delivered to thetumor site. To our knowledge, we present the first study ofsurface-engineered NK cells as well as the first study using CAR.NKcells for tumor-targeted drug delivery. We believe that ourCAR.NK-mediated drug delivery system can be expanded to include not onlythe delivery of traditional chemotherapeutic agents, but otheranticancer treatments such as immunomodulators and small molecules thataffect the tumor microenvironment.

Cancer immunotherapy has attracted much attention as an alternative oraddition to chemotherapy, and currently a few clinical trials are usingCAR-engineered T (CAR-T) cells to target patients with relapsed solidcancers, such as pancreatic, ovarian, prostate, and lung cancers.However, CAR-T therapy relies on the ex vivo expansion of the patient'sautologous T cells, which presents logistical issues and delays thestart of the treatment while cells are in preparation (typically 2-3weeks for the expansion of CAR-engineered immune cells for clinicaluse). These issues could be ameliorated in part by using an allogeniccell line instead of autologous cells; while there are few functionalcytotoxic T cell lines available, there are several functional, immortalNK cell lines. Of these NK cell lines, NK92 is the most promising andthe only NK cell line used in clinical trials.

There are a number of potential benefits to using CAR-engineered NK92cells over CAR-T cells. CAR-engineered NK92 cells may provide analternative “off-the-shelf” vehicle for CAR-based therapy as well asprovide more targeted drug delivery to the tumor site through surfaceengineering. NK92 cells double every 2-4 days, allowing for easyexpansion, modification, and storage under good manufacturing practice(GMP) conditions. NK92 cells are identical to the parental cell line,eliminating problems with donor variability. There would be no lag timerequired for the ex vivo expansion and modification of autologous immunecells, which is especially crucial in patients with aggressive cancers,where a treatment delay of days to weeks could impact outcome. NK92cells are safe to use clinically if irradiated, which preventsproliferation. This decreases the risk of off-target effects compared toCAR-T cells. Short-lived CAR-engineered NK92 cells can be treated as a“living drug”, redosing as necessary. Finally, allogenic NK92 cell-basedtherapies are less expensive than autologous CAR-T cell therapies—onegroup estimated that each CAR-T protocol costs upwards of $250,000 perpatient, but NK92 cells used in the clinic cost around $20,000 perpatient.

We have demonstrated that CAR.NK cells conjugated to PTX-loaded cMLVsoffer targeted drug delivery and improved antitumor efficacy. We believethat targeted drug delivery using surface-engineered CAR.NK cells iswidely applicable, as both the CAR target and the drug payloadpotentially can be altered to treat a variety of cancer types. Overall,this study shows a promising combination of immunotherapy and drugdelivery for enhanced antitumor treatment.

The various methods and techniques described above provide a number ofways to carry out the application. Of course, it is to be understoodthat not necessarily all objectives or advantages described can beachieved in accordance with any particular embodiment described herein.Thus, for example, those skilled in the art will recognize that themethods can be performed in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objectives or advantages as taught or suggested herein.A variety of alternatives are mentioned herein. It is to be understoodthat some preferred embodiments specifically include one, another, orseveral features, while others specifically exclude one, another, orseveral features, while still others mitigate a particular feature byinclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be employed invarious combinations by one of ordinary skill in this art to performmethods in accordance with the principles described herein. Among thevarious elements, features, and steps some will be specifically includedand others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the application extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses and modifications and equivalents thereof.

Preferred embodiments of this application are described herein,including the best mode known to the inventors for carrying out theapplication. Variations on those preferred embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. It is contemplated that skilled artisans canemploy such variations as appropriate, and the application can bepracticed otherwise than specifically described herein. Accordingly,many embodiments of this application include all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the application unless otherwise indicated herein orotherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that may have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

It is to be understood that the embodiments of the application disclosedherein are illustrative of the principles of the embodiments of theapplication. Other modifications that can be employed can be within thescope of the application. Thus, by way of example, but not oflimitation, alternative configurations of the embodiments of theapplication can be utilized in accordance with the teachings herein.Accordingly, embodiments of the present application are not limited tothat precisely as shown and described.

Various embodiments of the invention are described above in the DetailedDescription. While these descriptions directly describe the aboveembodiments, it is understood that those skilled in the art may conceivemodifications and/or variations to the specific embodiments shown anddescribed herein. Any such modifications or variations that fall withinthe purview of this description are intended to be included therein aswell. Unless specifically noted, it is the intention of the inventorsthat the words and phrases in the specification and claims be given theordinary and accustomed meanings to those of ordinary skill in theapplicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the invention to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe invention and its practical application and to enable others skilledin the art to utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this invention.

1. An engineered immune effector cell, comprising: a natural killer (NK)cell; and a plurality of nanoparticles bound to the surface of the NKcell; wherein the NK cell contains polynucleotides encoding one or morechimeric antigen receptors (CARs), the CAR comprising an extracellularantigen specific domain, and wherein the plurality of nanoparticlesencapsulates a chemotherapeutic agent in an amount that causes no orless than 15% cytotoxicity to a population of NK cells.
 2. A cellpopulation, comprising a plurality of the engineered immune effectorcell of claim 1, wherein each engineered immune effector cell comprises:a. a natural killer (NK) cell, further comprising polynucleotidesencoding one or more chimeric antigen receptors (CARs), the CARcomprising an extracellular antigen specific domain; and b. a pluralityof nanoparticles bound to the surface of the NK cell, at least onenanoparticle encapsulating a chemotherapeutic agent; and wherein thecell population has a total amount of the chemotherapeutic agent causingno or less than 15% cytotoxicity to the plurality of engineered immuneeffector cells, yet effective for inhibition or killing of tumor cells.3. The engineered immune effector cell of claim 1, wherein thenanoparticles are liposomes comprising crosslinked multilamellarliposomal vesicles (cMLVs).
 4. The engineered immune effector cell ofclaim 1, wherein the CAR binds to CD19.
 5. The engineered immuneeffector cell of claim 1, wherein the CAR binds Her2.
 6. The engineeredimmune effector cell of claim 1, wherein the CAR is a bispecific CAR andbinds CD19 and Her2.
 7. The engineered immune effector cell of claim 1,wherein the nanoparticles are cMLVs and the chemotherapeutic agent ispaclitaxel.
 8. The engineered immune effector cell of claim 1, whereinthe nanoparticles are cMLVs and the cMLVs are chemically bonded to thesurface of the NK cell at a number ratio between 100:1 and 150:1.
 9. Apharmaceutical composition comprising the engineered immune effectorcell of claim 1 and a pharmaceutically acceptable carrier.
 10. Apharmaceutical composition comprising the cell population of claim 2 anda pharmaceutically acceptable carrier.
 11. A method for treating asubject with a cancer comprising administering to the subject aneffective amount of the engineered immune effector cell of claim
 1. 12.A method for treating a subject with cancer comprising administering tothe subject a therapeutically effective amount of the pharmaceuticalcomposition of claim
 10. 13. The method of claim 12, wherein thepharmaceutical composition is administered via intravenous infusion. 14.The method of claim 12, wherein the subject has undergone total bodyirradiation (TBI), IgG1 antibody administration, or both, prior to theadministration of the pharmaceutical composition.
 15. The method ofclaim 12, further comprising one or more subsequent administrations ofthe pharmaceutical composition at weekly, biweekly, triweekly, monthly,quarterly or yearly intervals.
 16. The method of claim 12, wherein thesubject has one or more cancers selected from the group consisting ofleukemia, melanoma, renal cancer, prostate cancer, breast cancer, coloncancer, and lung cancer,